Les virus influenza de type A (IAV) appartiennent à la famille des Orthomyxoviridae. Ces virus enveloppés présentent un génome composé de 8 segments d’ARN simple brin, de polarité négative. Chaque segment est encapsidé par les nucléoprotéines (NP) et associé au complexe polymérase viral, hétérotrimère composé des sous-unités PB1, PB2 et PA, pour former la ribonucléoprotéine virale (RNPv). La protéine PB1 est la sous-unité catalytique responsable de l’activité ARN polymérase ARN-dépendante du complexe viral. La RNPv représente ainsi l’unité minimale de transcription et réplication du génome viral. En raison de la faible fidélité de la polymérase virale et l’absence d’activité de relecture, les IAV présentent un taux de mutation élevé, responsable du développement rapide de populations virales d’une grande diversité génétique, appelées quasi-espèces. Des études récentes ont permis d’identifier des mutants présentant une fidélité de réplication augmentée, due à des mutations uniques dans la sous-unité PB1. Comme décrit pour d’autres virus à ARN, différentes mutations peuvent avoir un effet similaire sur l’activité de la polymérase virale. Afin d’approfondir la caractérisation de la protéine PB1 nous avons recherché d’autres positions pouvant avoir un rôle dans la fidélité de la polymérase, la sélectivité des nucléotides ou la processivité du complexe. Pour cela, des banques de séquences PB1 mutées ont été générées par mutagénèse aléatoire pour deux sous-types de virus influenza A, H3N2 et H1N1pdm09, circulant actuellement chez l’homme. A partir de ces banques, des expériences de reconstitution transitoire de RNPv fonctionnelles (minigénome) en présence de ribavirine, un analogue nucléosidique mutagène, ont permis d’évaluer l’activité de la polymérase et de sélectionner, après subdivisions successives des banques, des mutations conférant une résistance au composé mutagène supérieure à celle de la polymérase sauvage. Les mutations ainsi identifiées dans différentes régions du segment PB1 ont ensuite été réintroduites de manière spécifique, par mutagénèse dirigée, dans la séquence du gène PB1. L’impact de ces mutations sur l’activité de la polymérase a été évalué par des expériences de minigénome en présence et absence de ribavirine. Les mutations pour lesquelles la résistance à la ribavirine a été confirmée ont alors été introduites par génétique inverse dans le contexte du génome viral complet. La majorité des mutations s’est avérée viable et a permis l’obtention de virus mutants infectieux. La capacité de multiplication des virus mutants a été évaluée en cellules MDCK et comparée à celle des virus sauvages correspondants, en absence et en présence de ribavirine. Ainsi, deux mutants porteurs de deux mutations différentes, localisées dans des régions distinctes de la protéine PB1, présentent une capacité à résister à la ribavirine supérieure à celle du virus sauvage. L’analyse de la diversité des populations virales, évaluée par séquençage à haut-débit, en utilisant la technologie Illumina, permettra de confirmer si cette résistance à la ribavirine est bien liée à une augmentation de la fidélité de la polymérase virale. Cette étude a ainsi permis de préciser les éléments de la protéine PB1 impliqués dans l’activité et potentiellement la fidélité de la réplication virale pour deux sous-types de virus influenza A / Influenza type A viruses (IAVs) belong to the Orthomyxoviridae family. The genome of these enveloped viruses consists of 8 single-stranded RNA segments of negative polarity. Each segment is encapsidated by oligomers of the nucleoprotein (NP) and associated with the viral polymerase complex, a heterotrimer composed of the PB1, PB2 and PA subunits to form the viral ribonucleoproteins (vRNPs). The PB1 protein is the catalytic subunit of the polymerase complex, harboring the RNA-dependent RNA polymerase activity. The vRNP represents the minimal functional unit for transcription and replication of the viral genome. Given the low fidelity and lack of proofreading activity of their polymerase, IAVs have a high mutation rate leading to the rapid development of viral populations with high genetic diversity, called quasispecies. Recent studies identified mutants with increased replication fidelity, due to single mutations in the PB1 subunit. As described with other RNA viruses, different mutations could have similar effects on the activity of the viral polymerase. To improve the characterization of the PB1 protein, we searched for other positions that may have a role on polymerase fidelity, nucleotide selectivity or complex processivity. For this purpose, random mutagenesis was used to generate libraries of mutated PB1 from influenza A virus subtypes H3N2 and H1N1pdm09, currently circulating in humans. From these libraries, transient reconstitution of functional vRNPs (minigenome) experiments were performed with ribavirin, a mutagenic nucleoside analog, to evaluate the polymerase activity. Upon selection based on the polymerase activity of successively subdivided libraries, PB1 mutations with increased polymerase activity in the presence of ribavirin relative to wild-type were identified in several regions of PB1. These mutations were specifically re-introduced in PB1 by directed mutagenesis. Their impact on polymerase activity was evaluated by minigenome experiments with and without ribavirin. Mutations with confirmed resistance against ribavirin were then introduced in the context of infectious virus by reverse genetics. Most corresponding mutant viruses could be rescued. Their growth characteristics were analysed in MDCK cells and compared to the corresponding wild-type viruses, in the presence or absence of ribavirin. Two mutants carrying two different mutations, located in distinct regions of the PB1 protein, displayed an improved capacity to resist ribavirin relative to the wild-type virus. Viral populations genetic diversity analysis by next-generation sequencing, using Illumina technology, will confirm whether the observed resistance against ribavirin is linked to an increase of the viral polymerase fidelity. This study provides insights into the PB1 domains involved in the activity and potentially the viral replication fidelity of two influenza A virus subtypes

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The influenza A virus genome codes for up to 12 proteins. Segment 2 encodes three proteins, the polymerase subunit PB1, a small protein PB1-F2 and an N-terminally truncated version of PB1 called N40. Different functions have been reported for PB1-F2 such as induction of apoptosis, regulation of the viral polymerase activity, enhancement of secondary bacterial infections and modulation of the innate immune system. So far, no function has been ascribed to N40. To study PB1-F2 in more detail, its coding sequence was deleted from its original position and inserted downstream of the PB1 (segment 2), NA (segment 6) or M (segment 7) open reading frames (ORF) employing different strategies, including the use of an overlapping Stop-Start cassette, a duplicated promoter sequence and the self-cleaving 2A peptide derived from foot-and-mouth disease virus. Viruses with bicistronic segments were rescued and tested for their ability to express PB1-F2. Whereas no expression of PB1-F2 was detected from bicistronic segments 2 and 7, expression of PB1-F2 from segment 6 was observed in high levels. However, the phenotype of all these viruses was similar to that of viruses lacking PB1-F2 which made mutational analysis of PB1-F2 not worthwhile. Previously, the function of PB1-F2 was mainly studied using a virus deficient in PB1-F2 production but showing increased N40 expression. In the present study, recombinant WSN viruses lacking either PB1-F2 or N40, or both proteins were engineered and the effects of these mutations on the viral life cycle were examined. Viruses deficient for PB1-F2 that overexpressed N40 showed the most attenuated phenotype, whereas the loss of PB1-F2 alone did not obviously affect virus replication. Reduced viral polymerase activity was observed for viruses lacking N40, however attenuation in vivo was only seen in combination with the loss of PB1-F2. Neither the loss of PB1-F2 nor N40 alone had a great impact, but changes in the expression level of both proteins were disadvantageous for the virus. Increased levels of N40 shifted the polymerase activity towards replication, suggesting a new function for N40. Thus, it was shown that the segment 2 gene products and their expression level influence viral replication and pathogenicity, and a careful design of mutant recombinant viruses is vital for determining the experimental outcome.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Influenza A is a negative stranded RNA virus with a segmented genome. Once the virus infects a cell it must replicate its full length viral genomic RNA (vRNA) through a positive sense complementary intermediate RNA (cRNA) as well as transcribe viral messenger RNA (mRNA) using the vRNA as a template. The regulation of whether the viral polymerase replicates the genome by synthesizing cRNA, or produces mRNA in order to make viral protein involves, the viral nucleoprotein (NP). We tried to find the sequence residues of NP that directly interact with the viral polymerase. We mutated to alanine several residues on NP that are surface exposed on recently solved crystal structures as well as those thought to be oriented toward the viral polymerase complex in cryo-EM studies. As a first screen, we tested these mutants in a mini-genome assay where the NP stimulation of the viral polymerase can be studied in transfected cells. Through this screen we found that the NP mutants that hindered its ability to stimulate polymerase activity the most were located in a loop between two alpha helixes in the head domain of NP located at residues 203 to 209. Specifically, the NP single mutants of R204, W207, and R208 were inactive in the mini-genome assay. Using RT-PCR we found that the cRNA to vRNA step of replication is severely inhibited by these mutations. Immunoprecipitation using transfected cells showed that the NP mutants lost the ability to bind all three polymerase subunits. This indicates that this loss of polymerase binding may be the reason the NP mutant fails to stimulate polymerase activity. To make sure that this loss of polymerase stimulation was not due to altering other functions of NP we made sure that the protein had proper cellular localization, oligomerization, and RNA binding abilities. Using immuniflourescence we found that mutant NP localized to the nucleus just like wild type. In order to test RNA binding and oligomerization we tested NP purified from a baculovirus expressing system. Using fluorescence polarization we found that NP binds single stranded RNA with similar affinity to wild type. Using gel filtration we found that mutant NP forms oligomers just like wild type. Using covariation analysis of how different positions in an amino acid alignment change relative to each other we predicted possible binding sites between NP and the three polymerase subunits PA, PB1 and PB2. Due to more complete crystal structure data we focused on the PA-NP interaction and found that covariation aided in finding binding sequence residues on PA but not NP. Another outcome of developing the covariation method was developing a program to view broad primary structure changes in large sequence alignments. This method has been informative in evaluating how amino acid positions in influenza have changed over time, as well as what defines specific residues as belonging to human or avian viruses. / text

Influenza A virus (IAV) causes annual seasonal epidemics and occasional pandemics resulting in significant levels of mortality and socio-economic costs worldwide. PB1-F2 is a small non-structural protein encoded by an alternate +1 open reading frame in the PB1 gene. PB1-F2 is considered to play important roles in primary influenza virus infection and post-influenza secondary bacterial pneumonia in mice. It is a multifunctional and enigmatic protein with diverse functions attributed to it and the precise contribution of PB1-F2 to the IAV life cycle in avian and mammalian hosts remains largely unknown. In the triple-reassortant H3N2 (TR H3N2) swine influenza virus (SIV) background, we found that PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology in pigs. On the other hand, in turkeys, deletion of PB1-F2 resulted in early induction of clinical disease and effective transmission among the turkey poults. Interestingly, the virulence associated 66S mutation in PB1-F2 abolished the ability of the IAV to successfully infect turkeys and transmit to in-contacts. These results highlight the strain- and species-specific role of PB1-F2 protein. We also demonstrated that specific amino acid residues in the C-terminal of PB1-F2 determine the pathogenicity of 2009 swine-origin pandemic H1N1 virus in a mouse model. The C-terminal residues 73K, 75R, and 79R together with 66S increased virus replication, decreased type I interferon response, increased infiltration of neutrophils and myeloperoxidase production in lungs resulting in acute respiratory distress syndrome (ARDS) in mice with characteristic clinical and pathological features of acute lung injury (ALI). Further, we found that PB1-F2 induces mitochondrial superoxide production and mitochondrial damage in a sequence dependent manner in IAV-infected lung epithelial cells. PB1-F2-mediated mitochondrial damage promotes Parkin-mediated mitophagy but suppresses the autophagic degradation of damaged mitochondria in the infected lung epithelial cells. Accumulated dysfunctional mitochondria likely to aggravate host cell death and inflammatory responses. Taken together, the present findings enhance our understanding of PB1-F2 protein as a virulence determinant in IAV infection in a species- and strain-specific manner and provide new insights into the impact of genetic changes in PB1-F2 on the host pathogenesis of virulent IAV strains. / Ph. D.

Unlike the binary compound, where the simple charge transfer between cation and anion, or the ternary compound, which is composed of two binary compound semiconductors with a common cation or anion and whose electronic structures usually can be derived from those of the two constituent binary compounds with some modifications, the electronic property of quaternary compound is quiet complicated and interesting because of its complex charge transfer due to the electronegativity differences of its composed atoms. In this thesis, the first-principles pseudofunction (PSF) method and the first-principles molecular dynamics (MD) method are used to investigate the complicated variations of the electronic properties of three kinds of quaternary compounds, namely titanates [Ba1-xSrxTiO3 (BSTO) and Pb1-xSrxTiO3 (PSTO)], manganites [La1-xSrxMnO3 (LSMO) and La1-xCaxMnO3 (LCMO)], and (SiC)1-x(AlN)x. First, for BSTO and PSTO titanates, the first-principles calculation results and O K-edge x-ray absorption near edge structure (XANES) measurements are used to study their electronic structures. Because the valence band maximum (VBM) and conduction band minimum (CBM) are composed of O-p and Ti-d partial densities of states (PDOS), respectively, the bowing upward of calculated band gaps are related to the bowing downward of the Ti-O bond lengths for both of BSTO and PSTO, though for PSTO Pb-p PDOS also contributes to states near CBM. The substitutions of Sr by Ba in BSTO and by Pb in PSTO are quiet different, and it is because Pb atom has two extra valence electrons and a larger electronegativity than other cations. Second, we provide a new interpretation of the insulator-like to metal-like and anti-ferromagnetic to ferromagnetic transitions with Sr and Ca doping concentrations of La1-xSrxMnO3 and La1-xCaxMnO3, which is based on the variations of the Sr and Ca induced delocalization of the Mn majority-spin eg subband and the lowering of the Mn minority-spin t2g subband down to the Fermi energy (EF). Moreover, this study also suggests that the magnetic properties of manganites result from a detailed balancing between the O-mediated super-exchange mechanism that favors anti-ferromagnetism and the delocalized-state mediated Mn-spin coupling that favors ferromagnetism. Third, for (SiC)1-x(AlN)x superlattice, where SiC and AlN layers arranged alternatively along a common c-axis, our analysis shows subtle charge transfer among Si, C, Al and N ions and the band gap is not linear but bows downwards with respect to x. The calculated results suggest that the direct band gap of (SiC)1-x(AlN)x can be tuned over a wide range from 2.97eV to 6.28eV. Thus, (SiC)1-x(AlN)x is potentially useful for optoelectronic applications. It can be inferred from the calculated electronic properties of the above three kinds of quaternary compounds, the subtle charge transfer is because of differing electronegativities of constituent atoms, especially cations, and the relative valence state of the dopant with respect to the host cation. The subtle charge transfer also influences the magnetic properties of these hole-doped manganites. Moreover, the quaternary compounds have four kinds of atoms with different electronegativities and relative orbital energies, the complicated competition and balancing between the occupation of orbitals and charge transfer render the electronic properties of these material unable to be predicted from constituent binary oxides/semiconductors or even ternary compounds. For example, even though Pb substitutes Sr only in Pb1-xSrxTiO3, the effective charges of Ti and O are significantly altered.

Lors de la phagocytose d'un micro-organisme, le complexe de la NADPH Oxydase des neutrophiles est activé. Il permet alors la production d'espèces réactives de l'oxygène qui contribuent à la destruction du pathogène. Ce complexe est constitué de facteurs cytosoliques (p67phox, p40phox, p47phox), d'une petite protéine G Rac et du flavocytochrome b558 membranaire, lui-même composé de p22phox et gp91phox. Des mutations dans les gènes codant pour certaines de ces protéines conduisent à une maladie génétique rare mais grave!: la granulomatose septique chronique (CGD). Au sein du complexe ternaire formé par les facteurs cytosoliques, il existe des interactions de type domaine SH3/motif polyproline et une interaction entre domaines PB1. Par Résonance Magnétique Nucléaire, nous avons caractérisé d'un point de vue structural l'interaction entre les domaines PB1 de p67phox et p40phox. Nous avons également étudié les conséquences de l'activation sur les interactions entre le motif polyproline C-terminal de p47phox et ses domaines SH3 partenaires. Ainsi, nous avons combiné l'analyse des structures des domaines SH3 de p40phox et SH3 C-terminal de p67phox, en complexe avec le polyproline C-terminal de p47phox, avec nos mesures d'affinité entre ces partenaires à différents stades de l'activation. Ces données ont été obtenues par fluorescence intrinsèque du tryptophane présent au sein des domaines SH3.

The development and characterization of experimental setups for angle-resolved photoelectron spectroscopy (ARPES) and spin- and angle-resolved photoelectron spectroscopy (SARPES) is described. Subsequently, the two techniques are applied to studies of the electronic band structure in topologically non-trivial materials. The laser-based ARPES setup works at a photon energy of 10.5 eV and a typical repetition rate in the range 200 kHz to 800 kHz. By using a time-of-flight electron energy analyzer electrons emitted from the sample within a solid angle of up to ±15 degrees can be collected and analyzed simultaneously. The SARPES setup is equipped with a traditional hemispherical electron energy analyzer in combination with a mini-Mott electron polarimeter. The system enables software-controlled switching between angle-resolved spin-integrated and spin-resolved measurements, thus providing the possibility to orient the sample by mapping out the electronic band structure using ARPES before performing spin-resolved measurements at selected points in the Brillouin zone. Thin films of the topological insulators (TIs) Bi2Se3, Bi2Te3 and Sb2Te3 are grown using e-beam evaporation and their surface states are observed by means of ARPES. By using a combination of low photon energies and cryogenic sample temperatures the topological states originating from both the vacuum interface (surface) and the substrate interface are observed in Bi2Se3 films and Bi2Se3/Bi2Te3 heterostructures, with total thicknesses in the ultra-thin limit (six to eight quintuple layers), grown on Bi-terminated Si(111) substrates. Band alignment between Si and Bi2Se3 at the interface creates a band bending through the films. The band bending is found to be independent of the Fermi level (EF) position in the bulk of the substrate, suggesting that the surface pinning of EF in the Si(111) substrate remains unaltered after deposition of the TI films. Therefore, the type and level of doping of the substrate does not show any large influence on the size of the band bending. Further, we provide experimental evidence for the realization of a topological crystalline insulator (TCI) phase in the narrow-band semiconductor Pb1−xSnxSe. The TCI phase exists for temperatures below the transition temperature Tc and is characterized by an inverted bulk band gap accompanied by the existence of non-gapped surface states crossing the band gap. Above Tc the material is in a topologically trivial phase where the surface states are gapped. Thus, when lowering the sample temperature across Tc a topological phase transition from a trivial insulator to a TCI is observed. SARPES studies indicate a helical spin structure of the surface states both in the topologically trivial and the TCI phase. / <p>QC 20130507</p>

The growing need of energy worldwide has lead to an increasing demand for alternative sources of power generation. Thermoelectric materials are one of the ‘green energy sources’ which convert directly heat into electricity, and vice–versa. The efficiency of this conversion is dependent on ‘figure of merit’ (z T), which depends on the material’s Seebeck coefficient (S), electrical resistivity (ρ) and thermal conductivity (κ) through the relation z T=S2T/ρκ, where T is the temperature. High values of z T lead to high efficiency, and therefore, z T must be maximized. Lead telluride is well–established thermoelectric material in the temperature range 350 K and 850 K. The aim of this thesis is to improve the z T of the material by adopting two different approaches – (i) doping/alloying and (ii) introducing additional interfaces in bulk i.e. having two phase PbTe. In this thesis, first an introduction about the thermoelectric phenomenon is given, along with the material parameters on which z T depends. A survey of literature associated with PbTe is done and the current status of thermoelectric devices is summarized briefly. This is followed by a description of the synthesis procedure and the measurement techniques adopted in this work. The first approach is the conventional alloying and doping of the material by which carrier concentration of the material is controlled so that maximum power factor Sρ2 is achieved and a simultaneous reduction of thermal conductivity takes place by mass fluctuation scattering. Under this, two systems have been studied. The first system is PbTe1−ySey alloys doped with In (nominal composition: Pb0.999In0.001Te1−ySey, y=0.01, 0.05, 0.10, 0.20, 0.25, 0.30). The compounds were single phase and polycrystalline. Lattice constants obtained from Rietveld refinement of X–ray diffraction (XRD) data showed that Vegard’s law was followed, indicating solid solution formation between PbTe and PbSe. Compositional analysis showed lower indium content than the nominal composition. Temperature dependent Seebeck coefficient showed all the samples to be n–type while Pisarenko plots showed that indium did not act as a resonant dopant. Electrical resistivity increased with temperature, while mobility vs T fitting showed a mixed scattering mechanism of acoustic phonon and ionized impurity scattering. Thermal conductivity followed a T1 dependence, which indicated acoustic phonon scattering. At high temperature, slight bipolar effect was observed, which showed the importance of control-ling carrier concentration for good thermoelectric properties. A z T of 0.66 was achieved at 800 K. The second alloy studied under this approach was Mn doped Pb1−ySnyTe alloy (nominal composition Pb0.96−yMn0.04SnyTe (y=0.56, 0.64, 0.72, 0.80)). All the samples followed Vegard’s law, showing formation of complete solid solution between PbTe and SnTe. Microstructure analysis showed grain size distribution of <1 µm to more than 10 µm. Seebeck coefficient showed all samples were p-type and the role of two valence band conduction in p–type PbTe based materials. Electrical resistivity showed a de-crease possibly due to (i) large carrier concentration or (ii) increased mobility due to Mn2+ ions. Thermal conductivity decreased systematically with decreasing Sn content. Bipolar effect was observed at high temperatures. Accordingly, the highest z T of 0.82 at 720 K was obtained for the sample with Sn (y=0.56) content due to optimum carrier concentration and maximum disorder. The second approach of having additional interfaces in bulk focuses on reducing thermal conductivity by scattering phonons. Under this approach, three systems were studied. The first system is PbTe with bismuth (Bi) secondary phase. The XRD and Ra-man studies showed that bismuth was not a dopant in PbTe, while micrographs showed micrometer–sized Bi secondary phase dispersed in bulk of PbTe. Reduction in Seebeck coefficient showed possible hole donation across PbTe–Bi interfaces, while electrical re-sistivity and thermal conductivity showed that the role of electrons at the interfaces was more important than phonons for the present bismuth concentrations. For the parent PbTe, z T of 0.8 at 725 K was reached, which, however decreased for bismuth added samples. The second system studied under the two phase approach was indium (In) added PbTe. Indium was not found to act as dopant in PbTe, while micrometer sized indium phase was found in PbTe bulk. A decrease in the electronic thermal conductivity ac-companied by a simultaneous increase of the electrical resistivity and Seebeck coefficient throughout the measurement range indicated increased scattering of electrons at PbTe-In interfaces. Higher values of the lattice thermal conductivity showed that the PbTe–In interfaces were ineffective at scattering phonons, which was initially expected due to the lattice mismatch between PbTe and In. For PbTe with 3 at. % In phase, z T value of 0.78 at 723 K was achieved. Under the two phase approach, as a comparative study, PbTe with both micrometer sized Bi and In phases together was prepared, in which no improvement in z T was found. A comparison of both the approaches showed that the alloying approach is better than the two–phase approach. This is because micrometer sized secondary phase scatter the electrons more than the phonons, leading to adverse effect on the transport coef-ficients, and hence, on z T. Alloying, on the other hand, is more beneficial in reducing thermal conductivity by mass fluctuation scattering, along with a simultaneous reduction of electrical resistivity.