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Influenza virus belongs to the Orthomyxovirus family of viruses that causes respiratory infection in humans, leading to morbidity and mortality. The mature influenza A virion has an envelope that contains two major surface glycoproteins proteins – hemagglutinin (HA) and neuraminidase (NA). HA is a highly antigenic molecules and is responsible binding to host cell surface receptors (Sialic acid), and membrane fusion between the viral membrane and the host endosomal membrane. Most of the antibody response generated against influenza virus either by vaccination or by natural infection is directed against HA. Influenza virus has segmented negative–sense RNA genome which gives the virus the ability to evade the host immune response by incorporating mutations (antigenic drift) and/or by reassotment with other subtypes of influenza A viruses (antigenic shift). Currently licensed vaccines which include an inactivated vaccine, a live attenuated vaccine, and recombinant subunit vaccine are beneficial for providing protection against seasonal influenza viruses that are closely related to the vaccine strain but fail to provide protection against drifted strains. This limits their breadth of protection and thus requires annual revaccination with reformulated vaccines. Also, because selection of a vaccine strain for the next season is purely based on surveillance and prediction, sometimes mismatches do happen between the selected vaccine strains and circulating viruses, resulting in a drastic decrease in vaccine efficacy and thus high morbidity and mortality. Furthermore, the production of these seasonal vaccines takes 6-8 months on an average, and does not guarantee protection against infection with novel reassortant viruses which can cause pandemics. To overcome the draw-backs of seasonal influenza virus vaccines and to enhance our pandemic preparedness, there is an increasing need for game-changing influenza virus vaccines that can confer robust, long-lasting protection against a broad spectrum of influenza virus isolates. Influenza hemagglutinin (HA) is highly immunogenic and thus a major target for vaccine design. HA is synthesized as a precursor polypeptide (HA0), assembles into a trimer, matures by proteolytic cleavage along the secretory pathway and is transported to the cell surface. Mature HA has a globular head domain, primarily composed of the HA1 subunit, which mediates receptor binding, while the stem domain, predominantly comprises of the HA2 subunit, and houses the fusion peptide. At neutral pH, the HA stem is trapped in a metastable state but undergoes an extensive conformational rearrangement at low pH in the late endosome (host-cell endosome) to trigger the fusion of virus and host membranes. Clusters of ‘antigenic sites’ have been identified in the head domain of HA, indicating that it harbors an almost continuous carpet of epitopes that are targeted by antibodies. However, these immunodominant sites constantly accumulate mutations to escape immune pressure, and thereby narrow the breadth of head-directed neutralizing antibodies (nAbs). In contrast to the highly-variable head domain, the membrane-proximal HA stem subdomain has much less sequence variability and, thus, is a desirable target for influenza vaccine development. In the recent past, several broadly neutralizing antibodies (bnAbs) targeting this subdomain with neutralizing activity against diverse influenza A virus subtypes have been isolated from infected people, further proving that this subdomain of HA can be targeted as a vaccine candidate. Steering the immune response towards this conserved, subimmunodominant stem subdomain in the presence of the variable immunodominant head domain of HA has been quite challenging. Alternatively, mimicking the epitome of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogenic capable of eliciting a broadly protective immune response has been difficult because of the metastable nature of HA. Addressing the aforementioned challenges, here we describe the design, stabilization and characterization of novel stem derived immunogens from HA of influenza A viruses using a protein minimization approach. Chapter 1 gives an overview of the influenza virus life cycle, nomenclature and classification of influenza virus; outlines the structural organization and functional properties of different viral proteins. An introduction to the kind of immune responses generated during vaccination or natural infection with the virus is discussed. The conventional vaccines that are currently used and their limitations, recent progress in the field of novel vaccine developmental approaches targeting the conserved epitopes on HA, is also described in this chapter. This chapter also gives a broad overview of bnAbs that have been isolated in the recent past, which target the novel antigenic signatures on HA. The design of a stem domain construct from an H3N2 virus (A/HK/68) is described in Chapter 2. In order to ensure that HA2 folds into the neutral pH conformation, regions of HA1 interacting with it were included in the design. Additionally, two Asp mutations were introduced in the B loop of HA2 to destabilize the low pH conformation and stabilize the desired native, neutral pH conformation. Studies using small peptides (57-98 of HA2) indicated that Asp mutations at positions 63 and 73 destabilized the low pH conformation. Studies on mutants with additional pairs of introduced Cys residues showed that the designed protein H3HA6 was folded into the neutral pH form. Immunization studies using mice showed that the protein was highly immunogenic and provided complete protection against a lethal dose of a homologous virus. Two constructs H3HA6a and H3HA6b, designed from the stem region of drifted H3N2 viruses (A/Phil/2/82 and A/Bris/10/07) were tested for protection against HK/68 to determine the extent of cross-strain protection provided by HA6. While HA6a (from A/Phil/2/82) provided near complete protection against HK/68, HA6b could protect against challenge only partially, possibly because of lower titers of antibodies elicited by this antigen. Studies using FcRγ chain knockout mice indicated that majority of the protection mediated by anti-HA6 antibodies was because of antibody mediated effectors functions, although neutralization as a mechanism of protection was also likely to contribute. In all the 18 subtypes of HA, the B loop contains residues that form the hydrophobic core of the extended coiled coil of the low pH form. As in the case of H3HA6, we suggest that these residues could be mutated to Asp to destabilize the low pH conformation. Two circularly permuted stem domain constructs from an H1N1 virus (A/PR/8/34) and an H5N1 virus (A/Viet/1203/04) were made. The design and characterization of these proteins is described in Chapter 3. H1HA6, H1HA0HA6 and H5HA6 were purified from inclusion bodies and refolded. The proteins H1HA6 and H1HA0HA6 were highly immunogenic and provided protection against a lethal challenge with homologous PR/8/34 virus. Anti-H1HA6 sera had higher titres of antibodies against heterogonous HAs as compared to convalescent sera. Stem derived immunogens from drifted H1N1 viruses (A/NC/20/99 and A/Cal/7/09) have been made and tested for cross-protection with PR/8/34 challenge. While H5HA6 also elicited high titers of antibodies, it could only protect partially against PR/8/34 challenge probably because high enough titers of cross-reactive protective antibodies were not elicited by this protein. These stem immunogens conferred robust subtype specific and modest heterosubtypic protection in vivo against lethal virus challenge. However, the immunogens, especially H1HA6, a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. The strategy used to improve the biophysical and biochemical properties and thus the immunogenicity of these stem derived immunogens is discussed in Chapter 4. A random mutagenesis library of H1HA6 was constructed by error prone PCR using modified nucleotide analogues. The library was displayed on the yeast cell surface to isolate mutants showing better surface expression and improvement in binding to the broadly neutralizing antibody CR6261 compared to the wild-type protein. We isolated few clones, of which one mutant (H1HA6P2) dominated the enriched population. The other mutants differed slightly from H1HA6P2. This mutant differs from the wild-type by two mutations K314E and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody foot-print (epitope). This mutant showed improved binding to CR6261 and exhibited significant improvement in surface expression. Improvement was also observed in binding of this mutant to F16v3-ScFv (another broadly neutralizing antibody). Two cysteine mutations were also introduced to further stabilize the trimeric form of the protein. Chapter 5 describes the biophysical and biochemical characterization of the high affinity isolated mutant at the protein level. We expressed this affinity matured mutant gene in E.coli and purified the protein from inclusion bodies. The stabilized mutant protein showed remarkable improvement in biophysical and biochemical properties and was recognized by stem directed conformation sensitive broadly neutralizing antibodies CR6261, F10 and F16v3 with affinity comparable to the full-length HA ectodomain. These results clearly suggest that this mutant protein is properly folded in its native pre-fusion conformation and thus can be an excellent candidate for eliciting stem directed broadly neutralizing antibodies. All these stabilized versions of stem derived immunogens will be tested for immunogenicity and cross-protection with different viral challenges. Chapter 6 describes the development of a method for mapping antibody epitopes (especially conformational epitopes) down to the residue level. Using a panel of single cysteine mutants, displayed on the yeast cell surface, this bypasses the need for laborious and time consuming protein purifications steps used in conventional methods for epitope mapping. We made a panel of single cysteine mutants, covering the entire surface of the antigen (CcdB, a bacterial toxin protein), displayed each mutant individually as well as in a pool, representing all mutants together on the yeast cell surface, and covalently labeled the cysteine with biotin-PEG2-maleimide to mask the area. The effect on antibody binding was monitored to identify the residues and relative positions important for antibody interactions with the displayed antigen by flow cytometry. By using this method we were able to map the conformational as well as linear epitopes of a panel of monoclonal antibodies down to the residue level with ease, and also identify the regions on the antigen which contribute to the antigen city during immunization in different animals. Since, this method is quite easy, rapid and gives in-depth information about antigenic epitopes, it can be useful in rational design of epitomes specific vaccines and other antibody therapeutics. It can easily be extended to other display systems and is a general approach to probe macromolecular interfaces.

Made available in DSpace on 2016-01-26T18:55:43Z (GMT). No. of bitstreams: 1 Claudia Lizandra Ricci.pdf: 291013 bytes, checksum: b9dccb042e091da74b7d28825ae3639d (MD5) Previous issue date: 2015-03-17 / The objective of this study was to measurement of the intraocular pressure (IOP) with Kowa HA-2 tonometer in cats analyzing the calibration, the accuracy and the validation of ambulatory clinical use. For calibration the post-mortem study was accomplished in 10 healthy eyes of 5 cats comparing the ocular manometry with the values of the IOP checked with the tonometer. For evaluation of the accuracy an in vivo study was accomplished in 20 healthy eyes of 10 anesthetized cats being compared the ocular manometry with the IOP obtained with the tonometer. For validation of the ambulatory clinical study of the IOP measurement was accomplished in 78 eyes of 39 healthy cats, in 7 eyes with clinical signs of glaucoma and in 20 eyes with clinical signs of uveitis. The correlation coefficient (r²) between the manometer and the tonometer was 0.993 and the equation of lineal regression was y=0.0915x+0.0878 in postmortem study. In the in vivo study the medium values of IOP in the manometry were 15.6±1.1 mmHg and in the tonometry were 15.5±1.2 mmHg, there was no statistics significant difference between the manometry and the tonometry. In the ambulatory clinical study with healthy cats the medium values of IOP with the tonometer were 15.0±1.5 mmHg, in the eyes with clinical signs of glaucoma were 38,4±8,1 mmHg and in the eyes with clinical signs of uveitis were of 10,4±2,0 mmHg. Therefore, there was a satisfactory correlation and accuracy between the IOP values obtained by direct ocular manometry and the tonometer in question. In the ambulatory clinical study the IOP values obtained with the tonometer were compatible for animals with healthy eyes and with clinical signs of glaucoma and uveitis. So, we can conclude that the Kowa HA-2 tonometer can be used in the routine ophthalmic examination, as it is a practical method for IOP measurement in cats. / O objetivo deste estudo foi mensurar a pressão intraocular (PIO) com o uso do tonômetro Kowa HA-2 em gatos, analisando a calibração, a acurácia e a validação do seu uso clínico ambulatorial. Para calibração foi realizado o estudo post-mortem em 10 olhos sadios de 5 gatos comparando a manometria ocular com os valores da PIO aferida com o tonômetro. Para avaliação de sua acurácia foi realizado um estudo in vivo em 20 olhos sadios de 10 gatos anestesiados comparando-se a manometria ocular com a PIO obtida com o tonômetro. Para validação do seu uso clínico ambulatorial foi realizado um estudo da mensuração da PIO em 78 olhos sadios de 39 gatos, em 7 olhos com sinais clínicos de glaucoma e em 20 olhos com sinais clínicos de uveíte. O coeficiente de correlação (r²) entre o manômetro e o tonômetro Kowa HA-2 foi de 0,993 e a equação de regressão linear foi y=0,0915x+0,0878 no estudo postmortem. No estudo in vivo, os valores médios de PIO na manometria foram de 15,6±1,1 mmHg e na tonometria foram de 15,5±1,2 mmHg não havendo diferença estatística significativa entre a manometria e a tonometria. No estudo ambulatorial com os gatos sadios, os valores médios de PIO com o tonômetro Kowa HA-2 foram 15,0±1,5 mmHg, nos olhos com sinais clínicos de glaucoma foram 38,4±8,1 mmHg e nos olhos com sinais clínicos de uveíte foram 10,4±2,0 mmHg. Houve, portanto, correlação e acurácia satisfatória entre os valores de PIO com a manometria e o tonômetro em questão. No estudo ambulatorial os valores de PIO obtidos com o tonômetro foram compatíveis para animais com olhos sadios e com sinais clínicos de glaucoma e uveíte. Desta maneira, podemos concluir que o tonômetro Kowa HA-2 pode ser empregado no exame oftálmico de rotina, pois trata-se de um método prático na aferição da PIO em gatos.

Made available in DSpace on 2016-07-18T17:53:15Z (GMT). No. of bitstreams: 1 Claudia Lizandra Ricci.pdf: 291013 bytes, checksum: b9dccb042e091da74b7d28825ae3639d (MD5) Previous issue date: 2015-03-17 / The objective of this study was to measurement of the intraocular pressure (IOP) with Kowa HA-2 tonometer in cats analyzing the calibration, the accuracy and the validation of ambulatory clinical use. For calibration the post-mortem study was accomplished in 10 healthy eyes of 5 cats comparing the ocular manometry with the values of the IOP checked with the tonometer. For evaluation of the accuracy an in vivo study was accomplished in 20 healthy eyes of 10 anesthetized cats being compared the ocular manometry with the IOP obtained with the tonometer. For validation of the ambulatory clinical study of the IOP measurement was accomplished in 78 eyes of 39 healthy cats, in 7 eyes with clinical signs of glaucoma and in 20 eyes with clinical signs of uveitis. The correlation coefficient (r²) between the manometer and the tonometer was 0.993 and the equation of lineal regression was y=0.0915x+0.0878 in postmortem study. In the in vivo study the medium values of IOP in the manometry were 15.6±1.1 mmHg and in the tonometry were 15.5±1.2 mmHg, there was no statistics significant difference between the manometry and the tonometry. In the ambulatory clinical study with healthy cats the medium values of IOP with the tonometer were 15.0±1.5 mmHg, in the eyes with clinical signs of glaucoma were 38,4±8,1 mmHg and in the eyes with clinical signs of uveitis were of 10,4±2,0 mmHg. Therefore, there was a satisfactory correlation and accuracy between the IOP values obtained by direct ocular manometry and the tonometer in question. In the ambulatory clinical study the IOP values obtained with the tonometer were compatible for animals with healthy eyes and with clinical signs of glaucoma and uveitis. So, we can conclude that the Kowa HA-2 tonometer can be used in the routine ophthalmic examination, as it is a practical method for IOP measurement in cats. / O objetivo deste estudo foi mensurar a pressão intraocular (PIO) com o uso do tonômetro Kowa HA-2 em gatos, analisando a calibração, a acurácia e a validação do seu uso clínico ambulatorial. Para calibração foi realizado o estudo post-mortem em 10 olhos sadios de 5 gatos comparando a manometria ocular com os valores da PIO aferida com o tonômetro. Para avaliação de sua acurácia foi realizado um estudo in vivo em 20 olhos sadios de 10 gatos anestesiados comparando-se a manometria ocular com a PIO obtida com o tonômetro. Para validação do seu uso clínico ambulatorial foi realizado um estudo da mensuração da PIO em 78 olhos sadios de 39 gatos, em 7 olhos com sinais clínicos de glaucoma e em 20 olhos com sinais clínicos de uveíte. O coeficiente de correlação (r²) entre o manômetro e o tonômetro Kowa HA-2 foi de 0,993 e a equação de regressão linear foi y=0,0915x+0,0878 no estudo postmortem. No estudo in vivo, os valores médios de PIO na manometria foram de 15,6±1,1 mmHg e na tonometria foram de 15,5±1,2 mmHg não havendo diferença estatística significativa entre a manometria e a tonometria. No estudo ambulatorial com os gatos sadios, os valores médios de PIO com o tonômetro Kowa HA-2 foram 15,0±1,5 mmHg, nos olhos com sinais clínicos de glaucoma foram 38,4±8,1 mmHg e nos olhos com sinais clínicos de uveíte foram 10,4±2,0 mmHg. Houve, portanto, correlação e acurácia satisfatória entre os valores de PIO com a manometria e o tonômetro em questão. No estudo ambulatorial os valores de PIO obtidos com o tonômetro foram compatíveis para animais com olhos sadios e com sinais clínicos de glaucoma e uveíte. Desta maneira, podemos concluir que o tonômetro Kowa HA-2 pode ser empregado no exame oftálmico de rotina, pois trata-se de um método prático na aferição da PIO em gatos.

Labour Migration of the Hawan Ha Clan Mongghul to Aksu in Xinjiang Abstract: The current work focuses on the ongoing labour migration of the Mongghul Ha Clan from Hawan, Gansu Province to Aksu in southern Xinjiang. It presents the motivation for the migration, and examines the social, economic and cultural changes the migration has catalyzed. Impacts on local Uyghur engendered by in-migrants are discussed. The work also features an overview and a generalization of the contemporary labour migration in China and Xinjiang in particular. Interviews with migrants are transcribed in Mongghul and translated to English and serve as an important source for the work.

Als Schlüsselenzym im Renin-Angiotensin System übernimmt das Angiotensin Converting Enzyme (ACE) eine wichtige Rolle bei der Blutdruckregulierung. ACE spaltet das biologisch inaktive Angiotensin I zum vasokonstriktorisch wirksamen Angiotensin II, was zu einem Anstieg des Blutdruckes führt. Tier- und Humanstudien zeigten, dass die Aufnahme bekannter, aus dem Proteinabbau stammender ACE-Inhibitoren eine Absenkung des Blutdruckes bewirkte. In der Lebensmittelindustrie finden Hydrolysate von Milchproteinen, im speziellen von Molkenproteinen, Einsatz in hypoallergenen (HA) Säuglingsnahrungen. Obwohl das Phänomen einer ACE-Inhibierung durch HA-Nahrungen in vitro in der Literatur bereits Erwähnung fand, existieren bislang keine Angaben zu einer potentiellen Wirkung in vivo. In der vorliegenden Arbeit konnte für kommerzielle hypoallergene (HA) Säuglingsnahrung eine sehr starke ACE-Hemmung in vitro zeigen (IC50-Werte zwischen 20 und 104 mg Protein/liter), welche die für fermentierte Sauermilchprodukte dokumentierte Wirkung bei weitem überstieg.] Mittels RP-HPLC und ESI-TOF-MS konnte neben zahlreichen bekannten Peptiden mit ACE-hemmendem Effekt erstmals das aus der Primärsequenz von -Lactalbumin freigesetzte Dipeptid Ile-Trp in den HA-Nahrungen identifiziert und quantifiziert werden. Ile-Trp ist der bislang potenteste in Lebensmitteln nachgewiesene ACE-Inhibitor (IC50 = 0,7µM). HA-Nahrungen zeigten auch ex vivo im Zellsystem (HUVECs) einen stark ACE-hemmenden Effekt. Aus diesem Grunde wurde ein möglicher Einfluss der HA-Nahrungen auf den Blutdruck spontan hypertensiver Ratten untersucht. Hierfür wurden die Tiere im Rahmen einer Fütterungsstudie über 14 Wochen mit standardisiertem Futter, welchem HA-Nahrung (Gruppe 1), konventionelle Säuglingsnahrung (Gruppe 2) bzw. der bekannte ACE-Inhibitor Captopril (Gruppe 3) zugesetzt war, gefüttert. Eine vierte Gruppe mit Standardfutter diente als Kontrolle. Der Blutdruck wurde am wachen Tier nichtinvasiv mittels tail-cuff-Methode gemessen. Der systolische Blutdruck sank bei Verabreichung der HA-Nahrung nach 7 Wochen signifikant um 21 ± 8 mmHg ab im Vergleich zur Kontrollgruppe bzw. den mit konventioneller Säuglingsnahrung gefütterten Tieren. Captopril führte zur einer Blutdrucksenkung um 30 ± 7 mmHg.

The main focus of this PhD project is the development and validation of a psychometric instrument for the measurement of individual dispositions towards rules and principles. Literature review and focus groups were used to generate insights into the reasons why individuals prefer rules and principles. On the basis of that review, an initial item pool was created covering the conceptual space of dispositions towards rules and principles. The final instrument consists of 10 items, 5 items each for the rules and principles subscales. The psychometric analysis suggested that it is valid and reliable. The instrument has sound predictive power and was able to significantly predict individuals’ behavioral intentions in relation to rules and principles across contexts. I found there were gender and ethnic differences in the relationship between dispositions towards rules and principles scores and behavioural intentions. This PhD is relevant to an emerging literature in behavioural accounting research that examines how practitioners’ personal characteristics and styles affect financial reporting practice.

This thesis aims to examine a number of issues related to exchange rate movements at different time horizons: long-run, in terms of investigating equilibrium real exchange rates; medium-run, in terms of investigating predictability of exchange rates in out-of-sample forecasting contexts; and short-run, in terms of studying high-frequency exchange rate dynamics in the actual foreign exchange trading. Specifically, we reassess four topics concerning exchange rate movements through macroeconomic fundamental analysis and microstructure approaches to exchange rates. With macro approaches, our study demonstrates, in a panel data setting, the link between real exchange rates and net foreign asset could be through the association between real exchange rates and trade balance. The panel study indicates the heterogeneity, in terms of the association between real exchange rates and trade balance, between the OECD economies and less mature economies. Our study on the monetary exchange rate model indicates the monetary model can describe the long-run behaviour of nominal exchange rates. Furthermore, we find the short-term exchange rate deviation adjustments to equilibrium and nonlinearities involved in the association between exchange rates and monetary fundamentals. With micro approaches, our study demonstrates, in short run, order flow has a significant impact on the contemporaneous exchange rate dynamics. However, we observe the prediction of order flow on the future exchange rate is quite weak. Our study also finds the weak interaction between macro news and private information in the exchange rate volatility study.

In this dissertation, I describe social, economic and political relations between Jews and Christians in medieval Europe before presenting the intellectual and religious context of Jewish life in Christian Spain. The purpose of this endeavor is to provide the framework for analyzing two works, one in Hebrew and one in Castilian, by the Spanish Jewish author Sem Tob de Carrión (1290- c.1370). Proverbios morales (1355-60), the Castilian text, is important to the Spanish literary canon because it is one of the first works of Semitic sapiential literature to be transmitted, in the vernacular, to a Christian public. However, it has generally been read by scholars of medieval Hispanic literature in isolation from his Hebrew writings. Given that Ma’aseh ha Rav (c. 1345) reveals essential aspects of his thought structure and intellectual milieu not found in Proverbios morales, it should be required reading for a thorough understanding of his worldview. In the Hebrew work, I draw parallels between the polemical language used by Sem Tob and historically documented ideological conflicts that took place among Jews in late medieval Spain and Provençe. Because it is written in a style that involves the weaving together of biblical quotations and allusions, the polemical language must be read in relation to the biblical contexts to which these allusions refer. When analyzed in this way, allegory pertaining to the ongoing dispute among Jews about philosophy and scriptural interpretation, and rebuttals of Christian truth claims, become apparent. Additionally, kabbalistic references and messianic allusions lend the work an esoteric character that sharply distinguishes it from Proverbios morales. This analysis of Ma’aseh ha Rav is used as a basis for comparing rabbinic and philosophical concepts that appear in both works. The general movement from opposition to unity that characterizes each text, and the ubiquitous “golden mean,” link the two works conceptually, and underscore Sem Tob’s preoccupation with harmonizing contradictions on both the spiritual and social levels of existence. This aspect of his thought reflects the general intellectual climate of his milieu, which is characterized by the blending, or intertwining, of the main doctrines of medieval Judaism--philosophy, mysticism, and Talmudic-traditionalism. / text