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Effective methods for isolation and purification of glycoproteins are of increasing significance to the rapidly growing biopharmaceutical and diagnostic industry. Glycoproteins represent the majority of therapeutic proteins on the market and are effectively used to treat immune disorders, infections, cancers, and other diseases. Targeting these glycoproteins is also critical to an emerging field of glycoproteomics aimed to understand structure-function relationships of glycans. Architecturally, these glycoproteins are proteins with covalently linked oligosaccharide chains of varying monosaccharide composition. Affinity chromatography has proven to be an excellent method of glycoprotein purification at the bench scale. However, chromatography in large scale production has its drawbacks. Column fowling, flowrate limitations, and diffusional constraints collectively hinder the effectiveness of the method. An alternative proposed in this dissertation is the use of affinity precipitation as a purification technique. The three main objectives are 1) develop and produce dual-functional, thermo-responsive affinity ligands from a biological host, 2) characterize and optimize the accompanying affinity precipitation method, and 3) apply the ligand and process to relevant, unmodified glycoproteins. The design of the thermo-responsive affinity construct was comprised of two main functional domains. The binding capability was achieved by selection of small ligands with affinity to a specific monosaccharide moiety. Two different lectins, or sugar binding proteins, were used in the fusion design: a fucose binding lectin from Ralstonia solanacearum, and a sialic acid binding lectin from Vibrio cholera. The thermo-responsive functionality was obtained by use of an elastin-like peptide (ELP), which confers inverse solubility relationship properties to the fusion construct. A small library of varying ELP chain lengths were designed to find the optimal size fusion for both production and function. These dual functional ligands were cloned and expressed in the microbial host, E. coli. Furthermore, secretion of these constructs was achieved by employing the Tat secretion pathway in combination with an outer membrane lipoprotein deletion mutant with a leaky periplasm phenotype. This secretory mechanism allows for easy isolation, avoidance of inclusion bodies, and no additional protease inhibitors. After successful production, the ligands were tested to confirm that dual functionality was preserved in fusion form. Once binding conditions and precipitation properties were ascertained, the purification ability was tested on model glycoproteins. Experimentation was carried out monitoring the purification yield, purity, and retained activity of the target enzymes. High contaminant solutions, such as cell lysates, were spiked with the model glycoproteins to mimic crude protein solutions. The purification ability of the constructs in these models was observed. The method was then implemented on two relevant glycoprotein applications: 1) purification of soybean peroxidase from a crude protein extract and 2) targeting the therapeutic protein erythropoietin from albumin rich, used CHO cell media. By implementation of the fucose targeting fusion construct, the unmodified soybean peroxidase is isolated from a natural crude extract from the soybean hull, a by-product of the soybean industry. The affinity precipitation method parameters were optimized with respect to ratios, temperatures, recycle, and elution buffers to achieve successful isolation of the low abundance enzyme. Under the optimized conditions, >95% recovery yield and a purification of 22.7 fold of an active, pure product was attainable. The purification of erythropoietin led to additional experimentation with high-abundant glycoprotein solutions, as well as expansion of the affinity ligand platform. The concept of multi-lectin affinity precipitation, using the fucose and sialic acid binding lection sequentially, was introduced and tested for purification capability. An industrially relevant scheme involving isolation of the erythropoietin from used CHO cell media allowed for an achievable yield of about 60%, with a resulting albumin depletion of about 85%. In addition to development of a pair of novel thermo-responsive affinity ligands for glycoprotein purification, this dissertation provides insight on possible improvements and future directions with respect to the thermo-responsive affinity ligand platform. This unique concept employs novel lectin fusions to target valuable glycoproteins using a method avoiding the major drawbacks associated with chromatography.

This thesis describes the development and use ofnon-immunoglobulin affinity proteins denoted affibodies asalternatives to antibodies in different immunotechnologyapplications. A 58 aa IgG Fc binding three-helix bundle domainZ, derived from staphylococcal protein A has been used asframework for library constructions, in which the face of themolecule involved in the native binding activity has beenengineered by combinatorial protein engineering. Recruting 13surface-located positions for simultanenous substitutionmutagenesis, using degenerated oligonucleotides for libraryassembly at the genetic level, two libraries differing in thechoice of codons were constructed to serve as general sourcesof novel affinity proteins. The libraries were adapted fordisplay onE. colifilamentous phage particles allowingin vitroselection of desired variants capable ofbinding a given target molecule. In selections using human IgAas target, several new IgA specific affibodies could beidentified. One variant ZIgA1, was further investigated and showed binding toboth IgA1 and IgA2 human subclasses as well as to secretoryIgA. This variant was further demonstrated uesful as ligand inaffinity chromatography purification for recovery of IgA fromdifferent samples including unconditioned human plasma.Affibodies of different specificities were also fused to otherprotein domains to construct fusion proteins of relevance forimmunotechnology applications. Using Fc of human IgG as genefusion partner, "artificial antbodies" could be produced inE. colias homodimeic proteins, where the antigenbinding was confered by N-terminally positioned affibodymoieties of different valencies. One area of application forthis type of constructs was demonstrated through specificdetection of the target protein by Western blotting. Exploitingthe uncomplicated structure of affibody affinity proteins, genefusions between affibodies and the homotetrameric reporterenzyme β-galactosidase were constructed, which could beproduced as soluble proteins intracellularly inE. coli. The potential use of such recombinantimmunoconjugates in immunotechnology was demonstrated in ELISAdot-blot and immunohistochemistry, where in the latter case IgAdepositions in the glomeruli of a human kidney biopsy could bespecfically detected with low background staining ofsurrounding tissues. In a novel format for sandwich ELISA, thepossible advantage of the bacterial origin of the affibodyclass of affinity proteins was investigated. As a means tocircumvent problems associated with the presence of humanheterophilic antibodies in serum, causing bakground signals dueto analyte-independent crosslinking of standard capture anddetection antibody reagents, assay formats based oncombinations of antibody and affibody reagents for capture anddetection were investigated and found to be of potentialuse. <b>Keywords:</b>phage display, combinatorial, affinity, IgAligand, immunohistochemistry, affibody-fusions

<p>This thesis describes the development and use ofnon-immunoglobulin affinity proteins denoted affibodies asalternatives to antibodies in different immunotechnologyapplications. A 58 aa IgG Fc binding three-helix bundle domainZ, derived from staphylococcal protein A has been used asframework for library constructions, in which the face of themolecule involved in the native binding activity has beenengineered by combinatorial protein engineering. Recruting 13surface-located positions for simultanenous substitutionmutagenesis, using degenerated oligonucleotides for libraryassembly at the genetic level, two libraries differing in thechoice of codons were constructed to serve as general sourcesof novel affinity proteins. The libraries were adapted fordisplay on<i>E. coli</i>filamentous phage particles allowing<i>in vitro</i>selection of desired variants capable ofbinding a given target molecule. In selections using human IgAas target, several new IgA specific affibodies could beidentified. One variant Z_IgA1, was further investigated and showed binding toboth IgA1 and IgA2 human subclasses as well as to secretoryIgA. This variant was further demonstrated uesful as ligand inaffinity chromatography purification for recovery of IgA fromdifferent samples including unconditioned human plasma.Affibodies of different specificities were also fused to otherprotein domains to construct fusion proteins of relevance forimmunotechnology applications. Using Fc of human IgG as genefusion partner, "artificial antbodies" could be produced in<i>E. coli</i>as homodimeic proteins, where the antigenbinding was confered by N-terminally positioned affibodymoieties of different valencies. One area of application forthis type of constructs was demonstrated through specificdetection of the target protein by Western blotting. Exploitingthe uncomplicated structure of affibody affinity proteins, genefusions between affibodies and the homotetrameric reporterenzyme β-galactosidase were constructed, which could beproduced as soluble proteins intracellularly in<i>E. coli</i>. The potential use of such recombinantimmunoconjugates in immunotechnology was demonstrated in ELISAdot-blot and immunohistochemistry, where in the latter case IgAdepositions in the glomeruli of a human kidney biopsy could bespecfically detected with low background staining ofsurrounding tissues. In a novel format for sandwich ELISA, thepossible advantage of the bacterial origin of the affibodyclass of affinity proteins was investigated. As a means tocircumvent problems associated with the presence of humanheterophilic antibodies in serum, causing bakground signals dueto analyte-independent crosslinking of standard capture anddetection antibody reagents, assay formats based oncombinations of antibody and affibody reagents for capture anddetection were investigated and found to be of potentialuse.</p><p><b>Keywords:</b>phage display, combinatorial, affinity, IgAligand, immunohistochemistry, affibody-fusions</p>

Cette thèse de doctorat comprend trois essais portant sur plusieurs sujets dans le domaine de la finance d'entreprise empirique. L'essai 1 étudie l'avantage informationnel des administrateurs indépendants ayant une expertise de l'industrie par rapport aux administrateurs indépendants sans cette expertise. Pour le faire, je regarde les bénéfices des transactions dites « d'initiés » réalisés par des administrateurs indépendants. Je trouve que les administrateurs indépendants possédant une expertise sectorielle obtiennent des bénéfices significativement plus élevés que les administrateurs indépendants sans ladite expertise. De plus, une augmentation de la proportion d'administrateurs indépendants possédant une expertise sectorielle pertinente au sein du conseil est associée à une meilleure performance de l'alliance, à une plus grande probabilité d'accomplissement des opérations de fusions et acquisitions et à une sensibilité moindre des investissements faites par l’entreprise aux prix de marché. Les résultats dans son ensemble suggèrent que les administrateurs experts de l'industrie ont une connaissance supérieure de l'entreprise et pourraient améliorer l'efficacité du conseil d’administration dans l'exercice de ses fonctions de surveillance et de conseil. L'essai 2 étudie l'effet préjudiciable de l'activité de vente à découvert sur les relations entre clients et fournisseurs. Nous montrons que le montant de ventes à découvert des actions d'un fournisseur est positivement associé à une probabilité plus élevée qu'un client important du fournisseur mette fin à la relation commerciale entre les deux. Une telle interruption peut être partiellement prévenue par la présence de détenteurs de « blocs » d’actions dans l'entreprise du fournisseur, particulièrement si ces détenteurs ont une orientation à long terme. Nos résultats sont conformes à l'hypothèse selon laquelle les vendeurs à découvert peuvent perturber les relations d'une entreprise avec des contreparties commerciales grâce à l'effet dit de « rétroaction », c’est-à-dire, le fait que les prix de marché affectent les décisions de l’entreprise. Par contre la présence de détenteurs de « blocs » constitue un mécanisme efficace pour contrer cette perturbation. L'essai 3 examine les raisons possibles du taux croissant d'annonces d'offres d’acquisition « groupées », dans lequel le management de l’acquéreur annonce une acquisition imminente le jour même où l’acquéreur déclare ses résultats trimestriels au marché. Cette pratique semble étrange étant donné que les offres groupées présentent des rendements d'annonce des acquéreurs nettement inférieurs, quel que soit le statut de la cible. Nos résultats pointent vers l'hypothèse de « révélation stratégique d’information » comme la raison la plus probable pour laquelle les entreprises choisissent de regrouper les annonces d'acquisition et les annonces de bénéfices. Ces acquéreurs, confrontés à des faibles bénéfices, dirigés par des PDG récemment nommés, et dans une situation où les analystes les contestent et où l'incertitude est grande, utilisent les annonces groupées comme un outil pour influencer la réception du marché sur leur performance. / This doctoral thesis includes three essays investigating several topics in the area of empirical corporate finance. Essay 1 studies the informational advantage of independent directors with industry expertise over independent directors without such expertise. To do so, I look at insider trading returns earned by independent directors. I find that independent directors with industry expertise earn significantly higher insider trading returns than do independent directors without such expertise. Moreover, an increase in the proportion of independent directors with relevant industry expertise on the board is associated with better alliance performance, a higher probability of M&A deal completion, and a lower investment-to-price sensitivity. Overall, the results suggest that industry expert directors have superior knowledge about the firm and could enhance board effectiveness in performing both monitoring and advisory roles. Essay 2 studies the detrimental effect of short selling activity on product market relationships. We show that higher supplier’s short interest is associated with a higher likelihood that a major client of the supplier terminates an existing relationship. Such interruption can be partially prevented by the presence of long-term blockholders in the supplier firm. Our results are consistent with the hypothesis that short sellers can disrupt a firm’s relationships with business counterparties through the feedback effect, but informed block ownership serves as an effective mechanism to counteract this disruption. Essay 3 investigates possible explanations for the increasing rate of “bundled” bid announcements, in which managers of the bidder announce an impending acquisition on the same day that the bidder reports its quarterly earnings to the market. This practice seems puzzling given that bundled bids exhibit significantly lower bidder announcement returns regardless of the target status. Our results point towards the strategic disclosure hypothesis as the most likely reason why firms choose to bundle acquisition and earnings announcements. Confronted with low earnings news, led by recently-nominated CEOs, in a situation in which they are challenged by analysts and uncertainty is high, bidders use bundled announcements as a tool to influence the market’s reception to the earnings.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19972号 / 薬科博第63号 / 新制||薬科||7(附属図書館) / 33068 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No / As this collection makes abundantly clear, the concept of “World Cinema” can be hard to define. To further establish a sensible definition of what “cult world cinema” might be is to stretch, contort and confuse understanding even further. Scholars have made bold attempts at defining what “cult cinema” might be that range from the “informal” to the “intertextual” by way of the “subcultural”. For instance, Karl and Philip French’s notion of cult as an “intense personal interest and devotion to a person, idea or activity” sought to access the devotional, sacramental aspect of engagement with cinema (French and French 1999: 6). A study by Jancovich and colleagues argued that it is the reception of films and their distinction from, and opposition to, the “mainstream” that defines films as “cult” (Janovich et al. 2003: 2). Mathijs and Sexton (2011) later promoted a rich, intertextual sense of what cult cinema might be, while admitting that because the numerous attempts at defining cult cinema approach the subject from the perspective of the vernacular—“highlighting elements that cannot be caught in a description”—any definition of cult cinema must be tantalisingly “intangible” and “intersubjective” (Mathijs and Sexton 2011: 6).

Les télomères sont des séquences d’ADN, généralement répétées en tandem, localisées à l’extrémité des chromosomes linéaires. Une des fonctions principales des télomères est de différencier l’extrémité des chromosomes des cassures double-brin, et ainsi de prévenir l’activation des voies de réparation de l’ADN. Chez les mammifères, cette fonction est plus spécifiquement assurée par le complexe shelterin. Il s’agit d’un complexe hétérogène composé de six protéines distinctes : TRF1, TRF2, POT1, RAP1, TPP1 et TIN2, qui interagit spécifiquement avec l’ADN télomérique. Au sein de ce complexe, les protéines RAP1 et TRF2 coopèrent afin d’empêcher l’extrémité des chromosomes d’être perçue comme un dommage de l’ADN, ce qui autrement aboutirait à des fusions inter-chromosomiques suite au processus de réparation. La protéine TRF2 se lie directement à la molécule d’ADN dans laquelle elle s’enroule de façon spécifique. Cette propriété est primordiale pour générer une structure d’ADN en forme de boucle, appelée t-loop, et dont le bon fonctionnement des télomères dépend. Les travaux effectués au cours de cette thèse ont mis en évidence deux scenarii indépendants dans lesquels la protéine RAP1 assure un rôle critique dans la stabilité des télomères. Premièrement, RAP1 peut prévenir les fusions inter-chromosomiques dans des cellules exprimant une forme altérée de TRF2 incapable de former des t-loops. Deuxièmement, l’inhibition de RAP1 dans des cellules en sénescence réplicative conduit à l’activation des voies de réparation de l’ADN et à la formation de fusions inter-chromosomiques. Ces observations font écho à des résultats précédents obtenus dans des cellules HeLa traitées avec l’inhibiteur de la télomérase BIBR1532, et dont l’expression de la protéine RAP1 était abolie par shRNA. De plus, j’ai montré que les fusions interchromosomiques engendrées par la perte de RAP1 sont dépendantes de la ligase IV, qui est un acteur principal de la voie de réparation de l’ADN par recombinaison non-homologue (NHEJ). Dans l’ensemble, ces travaux démontrent l’importance de la protéine RAP1 dans la stabilité des télomères lorsque la protéine TRF2 est non fonctionnelle, mais aussi dans des situations physiologiques telles que la sénescence réplicative. / In mammals, the shelterin complex is the guardian of telomere stability. It operates through a set of six proteins (TRF1, TRF2, POT1, RAP1, TPP1 and TIN2) that binds telomeric DNA and protects it from being recognized as DNA double-strand breaks and therefore control DNA repair and DNA damage response pathways. Among them, RAP1 and TRF2 cooperate and together protect chromosome extremities from end-to-end fusions. TRF2 is seen as a major factor to control telomere DNA topology by wrapping DNA around itself in a right handed manner. This property of TRF2 is required to promote the formation of t-loops, special DNA structures at telomeres that are considered as protective barriers to DNA damage response and fusion. Here we demonstrate two independent situations where RAP1 dysfunction is critical for telomere protection. First, in cells expressing a wrapping-deficient TRF2 allele that cannot form t-loops, RAP1 appears as a backup anti-fusion mechanism. Second, RAP1 downregulation in replicative senescent cells leads to telomere fusions and DNA damage response activation. This is consistent with similar observations in HeLa cells treated with the telomerase inhibitor BIBR1532, and in which RAP1 expression was abolished by an inducible shRNA system. In addition, we show that fusions triggered by RAP1 loss are dependent upon ligase IV, which is a key player of the classical non-homologous end-joining (c-NHEJ) repair pathway. Altogether, these results indicate that RAP1 takes over telomere protection when TRF2 cannot properly function or in the normal physiological situation, such as replicative senescence.

L’alternance de périodes glaciaires et interglaciaires durant les 20 derniers Ma a mené à des changements environnementaux répétés au niveau du plateau continental antarctique. C’est dans ce contexte que les téléostéens de la famille des Nototheniidae se sont adaptés et diversifiés à travers plusieurs vagues de radiations (dont les Trematominae), dominant l’Ichtyofaune australe. Parmi les Nototheniidae, le groupe « Trematomus » (genres Cryothenia, Pagothenia, Trematomus et Indonotothenia) est celui où l’on observe la plus grande diversité chromosomique, avec des nombres diploïdes de chromosomes allant de 24 à 58, impliquant de nombreux réarrangements ayant accompagné les spéciations. Nous avons cherché à caractériser ces remaniements chromosomiques. Avec un caryotype ancestral inféré de 2n = 48, une conservation des unités chromosomiques entre espèces, et une constance des tailles de génome, l’hypothèse de réarrangements structuraux sans polyploïdisation préalable est la plus probable. Afin de reconstruire l’histoire évolutive de ces événements, nous avons recherché les homologies chromosomiques interspécifiques. Ceci nous a permis de reconstituer les remaniements (majoritairement des fusions) que nous avons repositionnés sur la phylogénie résolue des « Trematomus ». Contrairement à ce qui a été publié pour le genre Notothenia, nos résultats suggèrent des acquisitions multiples et indépendantes. Les éléments transposables (ETs) peuvent être impliqués dans les remaniements chromosomiques par le biais de recombinaisons ectopiques. Ils participent alors à la diversification des lignées au cours de l’évolution. En raison de leur régulation épigénétique, leur mobilisation massive peut être induite en cas de variations environnementales importantes. Nous nous sommes intéressés à trois super-familles d’ETs (DIRS, Gypsy and Copia) dans ces génomes. Les DIRS1 ont montré des patrons d’insertions en points chauds dans les régions centromériques et péricentromériques. Etant donné leur mode de transposition décrit et leur propension à s’insérer dans des copies préexistantes, nous proposons un rôle des éléments DIRS1 comme facilitateurs des fusions observées lors de la diversification des « Trematomus ». / In the last 20 My, multiple glacial-interglacial cycles led to strong and repeated environmental changes on the Antarctic continental shelf. In this changing environment, nototheniid fishes diversified through several rounds of species radiation (one of which within Trematominae), and now constitute the dominant group in Antarctic teleosts. Among Nototheniidae, the group « Trematomus » (genera Cryothenia, Pagothenia, Trematomus and Indonotothenia) exhibits the highest chromosomal diversity, with diploid chromosome numbers ranging between 24 and 58, involving many rearrangements probably linked to speciation. We characterized the nature of these chromosomal repatternings. With an inferred ancestral state of 2n = 48 acrocentric chromosomes, a conserved number of chromosomal structural units, and a constancy of the genomes sizes we measured; the hypothesis of structural modifications is favored rather than a whole genome duplication associated to drastic reductions. In order to reconstruct an evolutionary scenario of such chromosomal rearrangements accompanying the trematomine diversification, we identified interspecific chromosomal homologies. This allowed us to reconstruct the rearrangements events (mostly centric and tandem fusions). We plotted them on a phylogeny we reconstructed based on our own ddRAD-seq data. Contrary to what was reported for the Notothenia, our results are in favor of independent acquisitions. Transposable elements (TEs) can lead to chromosomal rearrangements through ectopic recombination events, hinting at a role as drivers of specific-lineage diversification. Moreover, due to their epigenetic regulation, TEs can be mobilized when thermic changes occur. We focused on three retrotransposon superfamilies (DIRS, Gypsy and Copia) in nototheniid genomes. The DIRS1 showed unexpected accumulation patterns of insertion in the centromeric and pericentromeric regions. Given the mechanism of DIRS1 transposition and their tendency to sometimes insert on pre-existing copies (homing), we suggest a role of DIRS1 elements as facilitators of the fusions that occurred during the trematomine radiation.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.