Rozprawy doktorskie na temat „Viral phenomenon”

Endogenous viral elements, or viral genomic fossils, have proven extremely valuable in the study of the macroevolution of viruses, providing important, and otherwise unobtainable, insights into the ancient origin of viruses, and how their ancestors might have co-evolved with their hosts in the distant past. This type of investigation falls within the realm of paleovirology—the study of ancient viruses. Investigations of extant viruses and paleovirological analyses, however, often give conflicting results, especially those concerning viral evolutionary rates and timescales. Reconciling these two types of analyses is a necessary step towards a better understanding of the overall long-term evolutionary dynamics of viruses. The main study system of this thesis is foamy viruses (FVs). FVs are characterised by their stable co-speciation history with their hosts, allowing their evolutionary dynamics to be modelled and investigated over various timescales. This unique evolutionary feature makes FVs one of the best subjects for connecting recent and ancient viral evolution. The work here reports the discovery of several endogenous mammalian FVs, and examines how mammalian FVs co-evolve with their hosts. Analyses reveal a co-diversifying history of the two that could be dated back to the basal radiation of eutherians more than 100 million years ago. However, a small number of ancient FV cross-species transmissions could still be found, mostly involving New World monkey FVs. Based on this extended FV-mammal co-speciation pattern, this thesis investigates the long-term evolutionary rate dynamics of FVs, and shows that the rate estimates of FV evolution appear to decrease continuously as the rate measurement timescale increases, following a power-law decay function. The work presented here also shows that this so-called 'time-dependent rate phenomenon' is in fact a pervasive evolutionary feature of all viruses, and surprisingly, the rate estimates of evolution of all viruses seem to decay at the same speed, decreasing by approximately half for every 3-fold increase in the measurement timescale. Based on this power-law rate-decay pattern, we could infer evolutionary timescales of modern-day lentiviruses that are consistent with paleovirological analyses for the first time. Finally, this thesis reports the discovery of basal FV-like endogenous retroviruses (FLERVs) in amphibian and fish genomes. Phylogenetic analyses reveal that the progenitors of ray-finned fish FLERVs co-diversify broadly with their fish hosts, but also suggest that there might have been several ancient viral cross-class transmissions, involving lobe-finned fish, shark, and frog FLERVs. Again, by using the power-law rate-decay model, analyses in this thesis suggest that this major retroviral clade has an ancient Ordovician marine origin, originating together with their jawed vertebrate hosts more than 450 million years ago. This finding implies that the origin of retroviruses as a whole must be in the early Paleozoic Era, if not earlier. The results presented here bridge ancient and recent viral evolution.

La peste porcina clásica (PPC) es una enfermedad viral altamente contagiosa de cerdos domésticos y salvajes, incluida en la lista de enfermedades de declaración obligatoria a la Organización Mundial de Sanidad Animal. El agente causal, el virus PPC (VPPC), pertenece al género Pestivirus, familia Flaviviridae. A pesar de los grandes esfuerzos dirigidos a controlar y erradicar la PPC, continúa siendo una de las enfermedades más importantes para la sanidad animal y la industria porcina en todo el mundo. La circulación y la importancia intrínseca de cepas de VPPC de baja y moderada virulencia en las regiones endémicas ha sido objeto de discusión en los últimos años. Varios trabajos han demostrado el papel de cepas de baja virulencia en el nacimiento de lechones persistentemente infectados (PI) con el VPPC. No obstante, los mecanismos involucrados en este tipo de infecciones por VPPC no son bien conocidos, y los estudios existentes datan de hace 40 años. Por otro lado, la posible generación de persistencia viral después de la infección postnatal con VPPC era todavía una cuestión a resolver. Esta tesis demuestra la capacidad de cepas de VPPC de baja y moderada virulencia de inducir persistencia vírica de forma postnatal en cerdo doméstico y salvaje. Para ello, cerditos recién nacidos fueron inoculados de forma intranasal con el VPPC. Durante seis semanas, estos lechones permanecieron aparentemente sanos, a pesar de no generar respuesta inmunológica celular ni humoral específica para VPPC. Se demostró viremia permanente y alta carga de virus en todas las muestras de tejido, secreciones y excreciones de los animales persistentemente infectados (PI), hasta el fin del estudio. Además, se demostró la ineficacia de la vacunación de cerdos PI, de seis semanas de edad, los cuales no generaron respuesta de IFN de tipo I ni de tipo II, ni de anticuerpos durante 21 días tras la vacunación con una vacuna viva atenuada de VPPC (la C-strain). La falta de detección del ARN vacunal mediante RT-qPCR específica en sangre, excreciones y tejidos, incluyendo la tonsila, sugirió la ausencia de replicación del virus vacunal en los animales PI y un posible fenómeno de interferencia viral. Finalmente, jabalíes de seis semanas de vida con infección persistente de VPPC (primera infección) fueron inoculados con una cepa de alta virulencia de VPPC (segunda infección o superinfección). Los jabalíes PI no desarrollaron signos clínicos y mostraron alta carga del virus primario, causante de la persistencia, en todas las muestras analizadas. Por el contrario, el virus secundario no fue detectado ni por RT-qPCR ni por secuenciación, demostrando así el fenómeno de exclusión de la superinfección (ESI). La ausencia de una respuesta de IFN de tipo I y II y de anticuerpos respaldó los resultados previos de los estudios de esta tesis. Estos hallazgos demostraron por primera vez la capacidad del VPPC para inducir ESI in vivo, en cerdos con infección persistente. El fenómeno ESI probablemente explicaría la falta de respuesta de los animales con infección persistente después de la vacunación con la vacuna viva atenuada. Considerando la existencia de cepas de virulencia baja a moderada y su capacidad para producir formas de infección persistente, los resultados aquí presentados pueden tener potenciales repercusiones epidemiológicas, especialmente en el contexto endémico. Es importante destacar que los animales persistentemente infectados pasarían desapercibidos bajo control serológico, dada la ausencia de respuesta de anticuerpos específicos. Esta tesis doctoral ha contribuido a la comprensión de la patogénesis producida por el VPPC, que depende no sólo de la virulencia de la cepa sino también de la interacción virus-huésped, y abre nuevas líneas de investigación para comprender los mecanismos subyacentes que conducen a la generación de inmunotolerancia y persistencia del VPPC.
Classical swine fever (CSF) is a highly contagious viral disease of domestic and wild pigs, included in the list of diseases notifiable to the World Organisation for Animal Health. The causative agent, CSF virus (CSFV), belongs to the Pestivirus genus, Flaviviridae family. Over the last century, great efforts have been directed towards the control and eradication of CSF, which today remains one of the most important diseases for animal health and in the pig industry worldwide. The circulation and intrinsic importance of low and moderate virulence strains in the endemic regions has been extensively described. Also, it has been shown the role of these type of viral strains in the generation of “the pregnant carrier sow syndrome” and persistent infection of piglets after trans-placental transmission. However, the mechanisms involved in this form of CSFV infections are not well known, and the existing studies date over 40 years ago. By contrast, the possible generation of viral persistence after postnatal infection of pigs is still a question to resolve. Against this background, this thesis has demonstrated the capacity of low and moderate virulence CSFV strains to produce postnatal persistent infection early after birth. To this end, newborn piglets were inoculated intranasally with CSFV. During six weeks, these piglets remained apparently healthy, although they were not able to generate detectable CSFV specific humoral nor cellular immune responses, maintaining high virus load in blood, organs and body secretions and excretions. In addition, it has been demonstrated the ineffectiveness of vaccination of six-week-old PI pigs, which were unable to elicit a detectable innate immune response, in terms of IFN type-I production, as well as acquired immune responses (i.e. IFN type-II and antibodies) following vaccination with a CSFV live attenuated vaccine (C-strain). The RNA of the vaccine could not be detected by a specific RT-qPCR in any of the samples analysed after vaccination, including the tonsil, suggesting a superinfection exclusion (SIE) phenomenon between the persistently infecting virus (primary infection) and the CSFV vaccine strain (secondary infection). Finally, six-week-old wild boars with CSFV persistent infection (first infection) were inoculated with a CSFV strain of high virulence (second infection or superinfection). PI wild boars did not develop clinical signs and showed a high load of the primary virus, causing persistence, in all samples analysed. In contrast, the secondary virus was not detected by either RT-qPCR or sequence analyses, thus demonstrating the phenomenon of superinfection exclusion (SIE). The lack of innate and acquired immune responses supported the previous studies from this thesis. In addition, in vitro assays with the PBMCs isolated ex vivo from persistently infected animals, a well-known target for the CSFV viral replication, further corroborated the CSFV SIE phenomenon. These findings demonstrated for the first time the ability of CSFV to induce SIE in vivo, in swine with persistent infection. The SIE phenomenon would likely explain the unresponsiveness of animals with persistent infection after vaccination with the live attenuated vaccine. Considering the existence of CSFV strains of low and moderate virulence and their ability to produce persistent infection forms, the results showed here may have potential epidemiological repercussions, especially in the endemic context. Importantly, persistently infected animals would remain unnoticed under serological control, given the absence of specific antibody response. This doctoral thesis has contributed to the understanding of the pathogenesis of CSFV infection, which depends not only on the virulence of the strain but also on the virus-host interaction, and opens new lines of research to understand the immunological bases and of viral pathogenesis for the generation of immunotolerance and persistence, hitherto unknown.

This doctoral thesis commences with a meticulous examination on whether financial, social and institutional determinants associate with the migration performance in Europe and accordingly in Greece. Motivated by the intensity, the magnitude and the financial recession, this thesis presents three empirical chapters on the examination of the determinants that affect the phenomenon of migration. Prior to these three empirical studies a chapter introduces and defines all the variables used as well as the theoretical and methodological framework of the thesis. The first empirical chapter demonstrates a comprehensive sample of 15 European countries from 1990 – 2012, which have been divided into 3 groups (Weak-EMU, Strong-EMU and Non-EMU countries) in order to investigate the behaviour of each group during these periods. It follows a quantitative analysis of the economic and social determinants on migration, in order to comprehend their relationship with the phenomenon. The chapter concludes with the discussion of our results with an analytical review of the selected variables upon migration. Results reveal that Portugal, Ireland, Italy, Greece and Spain are countries that are more exposed to the financial crisis, something that consequently affects negatively the behaviour of each employed economic variable. Additionally, results detect that one significant outcome is that the GDP growth rate, inflation and the imports of goods are related to migration, while on the other hand the 10-year government bond yield is not affiliated to the phenomenon. The second empirical chapter covers the financial, social and institutional determinants that motivate Greek nationals to leave their country and emigrate to wealthier destinations during the recent financial crisis. First, it reviews the theoretical explanations for the efficiency of the factors on migration. It then provides a survey on the relevant empirical studies and subsequently an analysis of the variables, which have a significant impact on labour migration. Based on the theories presented, the study develops a model to explain how financial, social and institutional factors are correlated with the economic downturn and lead to adverse financial and social shocks such as massive migration outflows of Greek nationals. More precisely, the second chapter indicates that Greece has been very exposed to the financial crisis, thus had a strong impact on the decision of Greek natives to emigrate. As conditions deteriorate, Greece was in a severe financial situation due to the financial crisis and was dependent on the monetary policy support, something that emerged the country to experience major and drastic changes on its social cohesion. Further, we can identify, that debt to GDP, exports of goods, ln of imports of goods, long term unemployment and population growth are positively related to emigration from Greece, while on the other hand cash surplus, foreign direct investment and bank capital are negatively associated to the phenomenon. Finally, we employed advanced techniques to model the factors that motivate the existing regular immigrants in Greece to abandon the country and return to their own during the economic crisis period (return-migration). The findings reveal details on the imbalances of the economic, social and political framework of Greece that consequently affected negatively the growth rate of the country and created a fragile economy with high rates of unemployment and inflation. Hence, it compares the economic, social and institutional factors, which are related to the literature of return migration. According to the estimated results, the phenomenon of return migration is neither related in a predetermined way with unemployment rates nor poverty levels, but with tax revenue, corruption and government debt. Consequently, this situation had a strong negative impact in the behaviour of each financial, social and institutional determinant, with immediate result to the migrant families who pursue an improved quality of life back to their home country.

Theoretical studies of the molecular mechanisms responsible for the formation and stability of protein complexes have gained importance due to their practical applications in the understanding of the molecular basis of several diseases, in protein engineering and biotechnology. The objective of this project is to critically analyze and refine a coarse-grained force field for protein-protein interactions based on experimental thermodynamic properties and to apply it to cancer-related S100A4 protein system. Our ultimate goal is to generate knowledge for a better understanding of the physical mechanisms responsible for the association of particular proteins in different environments. We studied the role of short and long-range interactions on the complexation of homo-associations. Furthermore, we analyzed the influence of the pH and its correlation with the charge regulation mechanism. We analyzed and refined the adjustable Lennard-Jones parameter for a mesoscopic model based on experimental second virial data for lysozyme, chymotrypsinogen, and ribonuclease A via Monte Carlo simulations. From of that, the S100A3 protein was used to test the new calibrated parameters. Finally, we evaluated the dimerization process of S100A4 proteins, observing the role of physical-chemistry variables involved in the thermodynamical stability of different oligomers.
Estudos teóricos dos mecanismos moleculares responsáveis pela formação e estabilidade dos complexos de proteínas vêm ganhando importância devido às suas aplicações práticas no entendimento da base molecular de várias doenças, em engenharia de proteínas e biotecnologia. O objetivo deste projeto é analisar criticamente e aperfeiçoar um campo de força de granulidade grossa para interação proteína-proteína com base em propriedades termodinâmicas experimentais e aplicá-lo ao sistema proteico S100A4 relacionado com o câncer. Nosso objetivo final é gerar conhecimento para uma melhor compreensão dos mecanismos físicos responsáveis pelas associações de proteínas particulares em diferentes ambientes. Estudamos o papel das interações de curto e longo alcance na complexação de homo-associações. Além disso, analisamos a influência do pH e sua correlação com o mecanismo de regulação de cargas. Por meio de simulações Monte Carlo, analisamos e refinamos o parametro ajustável de Lennard-Jones para um modelo mesoscópico, usando dados experimentais do segundo virial para a lisozima, o quimotripsinogênio e a ribonuclease A. A partir disso, a proteína S100A3 foi usada para testar os novos parâmetros calibrados. Finalmente, foi avaliado o processo de dimerização das proteínas S100A4, observando o papel de algumas variáveis físico-químicas envolvidas na estabilidade termondinâmica de diferentes oligómeros.

P22 lysozyme, encoded by gene 19, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozymein its mode of action, substrate specificities, and in its structure. Gene 19 was located on the phage genome, subcloned, and then sequenced. lysozyme was produced in large quantities and purified for biochemical characterization and for crystallograpic studies. Gene 19consists of 146 codons, and encodes a protein with a molecular weight of 16,117. Amber mutations were created in gene 19 by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene 19 were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme, 20 are deleterious. The phage bearing amber mutations in gene 19that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced. A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 300C but not at 400C. When the triple change asn42->lys; ser46->leu; and ser43->pro is present the lysozyme produced is no longer temperature sensitive. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar, one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model. A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18->ser and ser23->lys. When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the original mutation.

The binding of HIV-1 Vif to the cellular cytidine deaminase Apobec3G and subsequent prevention of Apobec3G virion incorporation have recently been identified as critical steps for the successful completion of the HIV-1 viral life cycle. This interaction occurs in the cytoplasm where Vif complexes with Apobec3G and directs its degradation via the proteasome pathway or sequesters it away from the assembling virion, thereby preventing viral packaging of Apobec3G. While many recent studies have focused on several aspects of Vif interaction with Apobec3G, the subcellular localization of Vif and Apobec3G during the viral life cycle have not been fully considered. Inhibition of Apobec3G requires direct interaction of Vif with Apobec3G, which can only be achieved when both proteins are present in the same subcellular compartment. In this thesis, a unique approach was utilized to study the impact of Vif subcellular localization on Vif function. The question of whether localization could influence function was brought about during the course of studying a severely attenuated viral isolate from a long-term non-progressor who displayed a remarkable disease course. Initial observations indicated that this highly attenuated virus contained a mutant Vif protein that inhibited growth and replication. Upon further investigation, it was found that the Vif defect was atypical in that the mutant was fully functional in in vitro assays, but that it was aberrantly localized to the nucleus in the cell. This provided the basis for the study of Vif localization and its contribution to Vif function. In addition to the unique Vif mutant that was employed, while determining the localization and replication phenotypes of the differentially localized Vif proteins, a novel pathway for Vif function was defined. Copious publications have recently defined the mechanism for Vif inhibition of Apobec3G. Vif is able to recruit Apobec3G into a complex that is targeted for degradation by the proteasome. However, this directed degradation model did not fully explain the complete neutralization of Apobec3G observed in cell culture. Other recent works have proposed the existence of a second, complementary pathway for Vif function. This pathway is defined here as formation of an aggresome that prevents Apobec3G packaging by binding and sequestering Apobec3G in a perinuclear aggregate. This second mechanism is believed to work in parallel with the already defined directed degradation pathway to promote complete exclusion of Apobec3G from the virion. The data presented here provide insight into two areas of HIV research. First, the work on the naturally occurring Vif mutant isolated from a long-term non-progress or confirms the importance of Vif in in vivo pathogenesis and points to Vif as a potentially useful gene for manipulation in vaccine or therapy design due to its critical contributions to in vivo virus replication. Additionally, the work done to address the subcellular localization of Vif led to the proposal of a second pathway for Vif function. This could have implications in the field of basic Vif research in terms of completely understanding and defining the functions of Vif. Again, a more complete knowledge about Vif can help in the development of novel therapies aimed at disrupting Vif function and abrogating HIV-1 replication.

Since the first cases were reported over thirty years ago, great strides have been made to control disease progression in people living with HIV/AIDS. However, current estimates report that there are about 34 million individuals infected with HIV worldwide. Critical in the ongoing fight against this pandemic is the continuing development of highly active anti-retroviral therapies, ideally those with novel mechanisms of action. Currently, there are no medications approved for use that exploit the HIV-1 MA protein, despite its central role in multiple stages of the virus life cycle. This thesis sought to examine whether a highly conserved glutamate residue at position 99 in the understudied C-terminal helix of MA is required for HIV-1 replication. I characterized a panel of mutant viruses that contain different amino acid substitutions at this position using viral infectivity studies, virus-cell fusion assays, and immunoblotting. In doing so, I found that substitution of this glutamate with either a valine (E99V) or lysine (E99K) residue disrupted Env incorporation into nascent HIV particles, and abrogated their ability to fuse with target-cell membranes. In determining that the strain of HIV could affect the magnitude of E99V-associated defects, I identified a compensatory substitution at MA residue 84 that rescued both E99V- and E99K-associated impairments. I further characterized the MA E99V and E99K mutations by truncating HIV Env and pseudotyping with heterologous envelope proteins in an attempt to overcome the Env incorporation defect. Unexpectedly, I found that facilitating fusion at the plasma membrane was not sufficient to reverse the severe impairments in virus infectivity. Using quantitative PCR, I determined that an early post-entry step is disrupted in these particles that contain the E99V or E99K MA substitutions. However, allowing entry of mutant virus particles into cells through an endosomal route conferred a partial rescue in infectivity. As the characterization of this post-entry defect was limited by established virological methods, I designed a novel technique to analyze post-fusion events in retroviral infection. Thus, I present preliminary data regarding the development of a novel PCR-based assay that monitors trafficking of the viral reverse transcription complex (RTC) in an infected cell. The data presented in this thesis indicate that a single residue in MA, E99, has a previously unsuspected and key role in multiple facets of HIV-1 MA function. The pleiotropic defects that arise from specific substitutions of this amino acid implicate a hydrophobic pocket in MA in Env incorporation and an early post-entry function of the protein. These findings suggest that this understudied region of MA could be an important target in the development of a novel antiretroviral therapy.

Tolerance-based stem cell transplantation using sub-lethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. In order to study these two issues, we first established that mouse bone marrow recipients treated with sub-lethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. Our conditioning regimen of 6 Gy irradiation, a short course of anti-CD154 mAb and 25 million fully allogeneic BALB/c bone marrow cells consistently produced long-term, stable, and multilineage chimerism in C57BL/6 recipients. Furthermore, chimeric mice displayed donor-specific transplantation tolerance, as BALB/c skin allografts were permanently accepted while third-party CBA/JCr skin allografts were promptly rejected. We next determined both the safety and efficacy of this protocol by infecting chimeric mice with lymphocytic choriomeningitis virus (LCMV) either at the time of transplantation or at several time points afterwards. Infection with LCMV at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Surprisingly, infected allograft recipients also failed to clear the virus and died. Post-mortem study revealed hypoplastic bone marrow and spleens. Hypoplasia and death in these mice required the combination of 6 Gy irradiation, LCMV infection on the day of transplantation, and an allogeneic bone marrow transplant but did not require the presence of anti-CDl54 mAb. Allochimeric mice infected with LCMV 15 days after transplantation were able to survive and maintain their bone marrow graft, indicating that the deleterious effects of LCMV infection on host and graft survival are confined to a narrow window of time during the tolerization and transplantation process. The final section of this thesis studied the mechanisms of graft rejection and death in sublethally irradiated recipients of allogeneic bone marrow and infection with LCMV at the time of bone marrow transplantation. Infection of interferon-α/β receptor knockout mice at the time of transplantation prevented the engraftment of allogeneic bone marrow, but the mice survived. Therefore, IFN-αβ is involved in the development of marrow hypoplasia and death, whereas a second mechanism is involved in blocking the development of chimerism in these mice. Through the use of depleting mAb's and knockout mice we demonstrate that three types of recipients survived and became chimeric after being given sublethal irradiation, anti-CD154 mAb, an allogeneic bone marrow transplant and a day 0 LCMV infection: mice depleted of CD8+ T cells, CD8 knockout mice, and TCR-αβ knockout mice. Our data indicate that the mediator of bone marrow allograft destruction in LCMV-infected mice treated with costimulatory blockade is a radioresistant CD8+ NK1.1- TCRαβ+ T cell. We conclude that a non-cytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sub-lethally irradiated mice treated with costimulation blockade. The abrogation of allogeneic bone marrow engraftment is mediated by a population of CD8+ NK1.1- TCRαβ+ T cells and the mediator of hypoplasia and death is viral induction of IFN-αβ.

HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease. Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations. In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor. In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown. In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.

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Vem de longa data o interesse da ciência psicológica pela busca do autoconhecimento (James, 1890/1983). Neste contexto, os estudos oriundos do campo da autoconsciência, em especial com o advento da Teoria OSA (Duval & Wicklund, 1972), uma das primeiras teorias a considerar as discrepâncias entre o self e os padrões (Duval & Silva, 2001), têm reunido esforços no intuito de superar tamanha lacuna. Neste contexto, entusiasmado pela compreensão do self, em especial em sua vertente simbólica, bem como no instanciamento dos processos autoavaliativos por este operados, tramados à identificação da natureza dos padrões de atratividade e a forma como estes têm sido fenomenologicamente consciencizados no fluxo da experiência interna dos sujeitos, o presente estudo de tese objetiva identificar o que são os padrões de autoatratividade, qual sua dinâmica representacional no seio da experiência interna e o enlaçamento no processo autoavaliativo dos processos de atratividade autopercebida, autofocalização (autoconsciência situacional e disposicional), autoestima, bem estar (satisfação com a vida e felicidade), humor depressivo e religiosidade. Para o estudo em questão, foi recrutada uma amostra mista composta por 657 participantes (563 brasileiros e 94 portugueses), adolescentes, jovens, adultos e idosos, de ambos os sexos e orientações sexuais homossexual e heterossexual, residentes na Região Metropolitana do Recife e na cidade de Lisboa, Portugal. Os procedimentos relativos à coleta ocorreram em três etapas: a primeira com objetivo de levantar emicamente, por intermédio de procedimentos multimétodos, o campo semântico de autoatratividade (Estudo 1 – Qualitativo); a segunda, com base nos achados da etapa anterior, corresponde ao processo de elaboração e validação da Escala de Autoatratividade - EAA junto à pesquisa desta com o conjunto de instrumentos utilizados (Escala de Apreciação Corporal (EAC); Escala de Autoconsciência Situacional (EAS); Escala de Autoconsciência Disposicional (EAD); Escala de Autoestima de Rosenberg; Escala de Satisfação com a Vida; Escala de Felicidade Subjetiva; Escala CES-D (Rastreamento de depressão); Escala de Religiosidade Global (ERG) e Escala de religiosidade de item único, bem como o roteiro de entrevista Fenomenológico-Cognitiva dos Estados Autoconscientes – EFEA), a fim de permitir a identificação da multidimensionalidade da maquinaria psíquica atrelada ao construto atratividade dentre a amostra recrutada (Estudo 2 – Ex-post-facto) e a terceira etapa refere-se ao aprofundamento dos achados da etapa que a antecede, por intermédio da apresentação do gradiente fenomenal dos padrões de atratividade com base na identificação dos modos como estes são representados no fluxo da experiência dos participantes durante estado autoconsciente relacionado a autoatratividade (Estudo 3 – Fenomenal). A hipótese geral que dá sustentação ao estudo em questão considera que quanto mais autoconscientes e de modo reflexivo as pessoas são maior seria a capacidade que estas têm de minorar as influências que os padrões de atratividade exercem sobre os processos autoavaliativos, ocasionando prejuízos ao funcionamento psicológico saudável, além de que, os padrões, caso emerjam à consciência, no seio da experiência interna, se realizarão cognitivamente em elementos variados de natureza representacional, em especial na forma da fala interna e visualização interna. De modo específico, dentre o conjunto de hipóteses que dá sustenção ao referido estudo, destaca-se aquela que chama atenção ao papel moderador da religiosidade no possível impacto deletério que os padrões de atratividade ocasionam à vida das pessoas, haja vista, a importância e centralidade de tal dimensão para subjetividade humana. O modelo de análise de dados adotado no presente estudo de tese prezou, no tocante ao material quantitativo, pela investigação da dimensionalidade das escalas por via da metodologia das facetas (Guttman, 1968), com base nas Análises Multidimensionais não-métricas do tipo SSA (Similarity Structure Analysis, ver Guttman, 1968; Roazzi, 1995). Por sua vez, o material qualitativo foi submetido à análise de conteúdo (Estudo 1, Bardin, 1970) e a metodologia fenomenológica padrão (Estudo 2, Cott & Rock, 2008). Os principais achados do presente estudo de tese apontam que os padrões de autoatratividade e sua fenomenologia são representados cognitivamente na consciência por intermédio dos elementos da fala interna e das visualizações internas, associadas aos elementos do sentimento, bem como a consciência sensória. Não se encontrou, todavia, ocorrência de pensamento não simbolizado na análise fenomenal operada. A estrutura de tal padrão se organiza a partir de duas dimensões, uma física e outra não física, representadas pelos fatores: sensualidade, moralidade, apresentação pessoal, afetividade, inteligência, bom humor e asseio. Tais fatores quando correlacionados entre si, e junto as demais medidas utilizadas, apontam a presença de correlações estatisticamente significantes, em especial, no tocante às variáveis: Orientação sexual, no âmbito geral, os heterossexuais encontram-se mais próximos de um campo de afeto positivo, e os homossexuais nas cercanias do afeto negativo; Idade, os mais jovens, também situados num campo do afeto negativo, passando por um campo intermediário, onde há a presença de jovens adultos, findando com os participantes com idades entre 36 e 76 anos, cravados num campo de afeto positivo; Religiosidade, igualmente caracterizada por uma disposição polar, nas cercanias desta variável, os que se dizem religiosos, os heterossexuais e os adultos e idosos, e distanciando-se da religiosidade, localizados em plano antagônico, os que relatam não professar nenhuma fé, os adolescentes de 14 a 20 anos e os jovens homossexuais; e Nacionalidade, os portugueses mais próximos de um padrão de atratividade com base em princípios morais e os brasileiros simpáticos às variáveis sociodemográficas de natureza religiosa, inferindo que estas exercem influência sobre o instanciamento dos processos autofocalizadores, correlações estas cujos significados são corroborados pelos achados oriundos do estudo fenomenal. Tal estudo representa um esforço em dar visibilidade a uma temática pouco investigada na ciência psicológica, a natureza dos padrões de correção e o impacto destes nos processos autofocalizadores, em diálogo com uma perspectiva de mente dual, que contempla aspectos psicológicos e fenomenais da subjetividade e cognição humanas, a fim de contribuir com uma compreensão mais profunda sobre os aspectos cognitivos cruciais relacionados ao instanciamento de modos mais adaptados e, consequentemente, menos comprometedores da existência significativa e feliz.
The interest of the psychological science in the search for self-knowledge has come a long way (James, 1890/1983). In this context, studies from the self-awareness field, especially with the advent of the OSA Theory (Duval & Wicklund, 1972), one of the first to consider the discrepancies between the self and the patterns (Duval & Silva, 2001), have gathered efforts in order to overcome such gap. Within this framework, keen on understanding the self, especially in its symbolic aspect as well as in the instantiation of the self-assessment processes it operates, hatched in the identification of the nature of attractiveness patterns and how they have phenomenologically been made aware in the flow of the subjects‟ inner experience, this study aims to identify self-attractiveness patterns, their representational dynamics within the inner experience and the intertwining of the self-perceived attractiveness processes, of self-focusing (situational and dispositional self-awareness), self-esteem and well-being (life satisfaction and happiness), depressive mood and religiosity in the self-assessment process. For this study, a mixed sample of 657 participants (563 Brazilian and 94 Portuguese) was recruited, among whom were teenagers, adults and elderly people of both sexes, as well as homo and heterosexual orientations, living in the metropolitan area of Recife and in Lisbon, Portugal. The collecting-related procedures were carried out in three steps: the first one, to emically raise, through multimethod procedures, the semantic field of self-attractiveness (Study 1 - Qualitative); the second, based on the findings from the previous step, corresponds to the process of development and validation of the Self-attractiveness Scale – SAS, along with its survey with the set of instruments used (Body Assessment Scale - BAS); Situational Self-Awareness Scale (SSAE); Dispositional Self-Awareness Scale (DSAS); Rosenberg Self-Esteem Scale; Life Satisfaction Scale; Subjective Happiness Scale; CES-D Scale (Depression Tracking); Global Religiosity Scale (GRS) and Single-item Religiosity Scale, as well as the Self-Aware States Phenomenological-Cognitive interview script – SASPG), to enable the identification of the multidimensionality of the psychic machinery linked to the attractiveness construct within the recruited sample (Study 2 - Ex-post-facto). The third stage refers to deepening the findings of the previous one through the presentation of the phenomenal gradient of attractiveness patterns based on the identification of the ways they are represented in the flow of the participants‟ experience during the self-aware state related to self-attractiveness (Study 3 - Phenomenal). The general hypothesis that supports the present study considers that the more self-aware and reflective people are, the greater the capacity they have to reduce the influence attractiveness patterns have on self-assessment processes, which bring harm to healthy psychological functioning. Moreover, those patterns, should they emerge to awareness within the inner experience, will cognitively take place in a range of elements of representational nature, especially in the form of inner speech and inner visualization. In particular, among the set of assumptions that support this study, there is one that draws attention to the moderating role of religiosity in the possible deleterious impact attractiveness patterns cause to people's lives, given the importance and centrality of such dimension to human subjectivity. With regard to the quantitative material, the data analysis model adopted in this thesis study valued the investigation of the scale dimensionality by means of the facet methodology (Guttman, 1968), based on the non-metric SSA (Similarity Structure Analysis, see Guttman, 1968; Roazzi, 1995) type Multidimensional Analysis. In turn, the qualitative material was submitted to content analysis (Study 1, Bardin, 1970) and to the pattern phenomenological methodology (Study 2, Cott & Rock, 2008). The main findings of the present study indicate the self-attractiveness patterns and their phenomenology to be represented cognitively in the awareness by means of the inner speech and inner visualization elements, associated with both feelings and sensory awareness. There was no occurrence of non-symbolized thought in the carried out phenomenal analysis, though. The structure of such a pattern is set considering two dimensions, physical and non-physical, represented by the following factors: sensuality, morality, personal presentation, affection, intelligence, good humor and neatness. Such factors, when correlated and with the use of other measures, suggest the presence of statistically significant correlations, especially regarding the variables: Sexual orientation, in general, heterosexuals are closer to a positive affection ground, while homosexuals, around negative affection; Age, the youngest also being in a negative affection ground, passing to a middle ground where young adults are present, ending with between 36 and 76 year-old participants, nailed to a positive affection ground; Religiosity, also characterized by a polar layout around this variable, those who claim to be religious, the heterosexual, the adults and the elderly, as opposed to those distant from religiosity, located in an antagonistic plan, who report not to profess any faith, 14 to 20 year-old adolescents and young homosexuals; and Nationality, with the Portuguese closer to an attractiveness pattern based on moral principles, whilst the Brazilians sympathize with the sociodemographic variables of a religious nature, inferring that they influence the instancing of self-focusing processes, correlations whose meanings are corroborated by findings of the phenomenal study. This study represents an effort to give visibility to a little investigated topic in the psychological science, the nature of correction patterns and their impact on the self-focusing processes, dialoguing with a dual mind perspective, which includes psychological and phenomenal aspects of human subjectivity and cognition in order to contribute to a deeper understanding of the crucial cognitive aspects related to the instancing of more adapted and consequently less compromising forms of a meaningful and happy existence.

The bovine papillomavirus type 1 E2 protein is a multifunctional early viral protein with roles in all phases of the cell cycle. E2 is required during G1 as a transcription factor, in S phase to initiate viral replication and during mitosis to tether the viral genome to dividing DNA. The viral genome contains 17 E2 binding sites, the majority of which are concentrated in the long control region (LCR), a regulatory region that is upstream of the viral coding sequence. The role of these binding sites has been explored in vitro using small plasmids and E1 and E2 proteins expressed in bacteria and insect cells. In this study we attempt to examine the placement of E2 on its binding sites during all phases of the cell cycle and in the context of a stably replicating viral system. As part of the examination of the role of E2 during mitosis, we have also examined the role of the cohesin protein Scc1 in viral tethering. Two groups have published disparate reports identifying the cellular protein that binds to the transactivation domain of E2 to stably maintain viral genomes during cell division. Our group has published that it is the DNA helicase ChlR1 that is required for viral tethering, while it has been reported that it is the bromodomain protein Brd4 that is responsible. In this study we contribute to a report that shows that the cellular protein Scc1 binds to the viral genome through a ChlR1 independent mechanism. The cohesin protein binds to BPV-1 E2 at intermittent stages of the cell cycle and may be a factor in viral genome tethering. This interaction may also be important for regulating viral transcription.

The Human Immunodeficiency Virus (HIV) is able to infect CD4+ T cells as well as macrophages. Macrophage-tropism has been linked to determinants in the envelope of HIV. These determinants allow envelopes to exploit low levels of CD4 for infection. Macrophages are an important reservoir of virus, especially during chronic infection, and are likely responsible for the bulk of virus produced after CD4+T cells have declined. Viral factors that may impact the ability to infect macrophages are worth studying because this cell type is so important in infection. It was previously reported that the macrophage-tropic primary isolate AD8 was vpu-independent. The molecular clone YU-2, derived from brain tissue without culture, was also reported to be macrophage-tropic despite having a mutation in the vpu start codon. It was therefore possible that vpu-independent envelopes could evolve in vivo. To examine this possibility, I constructed chimeras containing wild type or defective vpu start codons, and gp160 sequences from AD8, YU-2 or SF162 (a vpu-dependent control). I also used full length AD8 and YU-2 with wild type or defective vpu start codons. I infected macrophages with equal amounts of virus, and measured viral output over two weeks. Viruses with defective vpu start codons were released to lower levels compared to their wild type vpucounterparts. In contrast to previous reports, the AD8 envelope is not vpu-independent for replication in macrophages. The YU-2 envelope is also not vpu-independent. Macrophage-tropic envelopes from late stages of infection can be sensitive to antibodies that bind the CD4 binding site on gp120, implying that macrophage-tropic envelopes have more exposed CD4 binding sites. Neutralizing antibodies may act as modulators of macrophage-tropism over the course of infection. Using chimeras containing gp120 sequences derived from the PBMC of four HIV+patients, I examined the capacity for envelopes to infect macrophages. Three patients (MM1, 4, and 8) had macrophage-tropic envelopes before and after developing autologous neutralizing antibodies. Three patients (MM1, 4, and 23) developed heterologous antibodies against IIIB, an easily neutralized T-cell line adapted strain of HIV-1. This data indicates that macrophage-tropism in these patients is not modulated by the presence of neutralizing antibodies. The macrophage-tropism of envelopes tends to segregate depending on the tissue origin of the virus. Envelopes from two separate tissues from the same patient exhibit very different infectivity characteristics. The B33 envelope, from brain tissue, is very infectious and is macrophage-tropic, while the LN40 envelope, from lymph node tissue, is weakly infectious and is not macrophage-tropic. Replacing the entire gp41 of LN40 with that of B33 restores some infectivity to LN40. The cytoplasmic domain of gp41 contains many motifs important for assembly and infectivity. To examine which motifs are responsible for the weak infectivity of LN40, I made chimeras of gp41, as well as point mutations in gp41. The LN40 chimera containing the entire gp41 of B33 restored the most infectivity. Point mutations in the palmitoylation site, Pr55gagbinding region, and dileucine motif at the C-terminus also restored infectivity when combined. Determinants in the gp41 cytoplasmic domain are responsible for the weak infectivity of LN40; however, it is possible that there are contributing determinants in gp120, such as the ability to use low levels of CD4. Here, I examined how changes in the vpu and env genes of HIV-1 can impact infectivity, especially infectivity of macrophages. Changes that adversely impact the virus’ ability to infect macrophages may also impact the overall course of disease. However, the data here show that retaining the ability to infect, and replicate in, macrophages give HIV an advantage. I speculate that retaining the ability to infect macrophages gives the virus a reservoir for later in disease, when CD4+ T cells have been depleted, as well as way of avoiding neutralizing antibodies. This work further defines the importance of macrophages in HIV-1 infectivity and disease.

The fusion protein of the Newcastle Disease Virus (NDV) contains three hydrophobic domains. To explore the topogenic signals of these domains, mutants were constructed in which each of the hydrophobic domains was deleted. The membrane insertion and topology of these proteins was characterized in a wheat germ cell-free translation system supplemented with canine microsomal membranes. The results indicated that the first 13 amino acids of the fusion protein are necessary to confer translation inhibition by SRP. Translocation of the nascent chains containing all or part of the first hydrophobic sequence resulted in the appearance of a species of higher molecular weight consistent with glycosylation of at least four of the five potential N-linked glycosylation sites. When glycosylation was inhibited with a glycosylation competitor peptide, signal sequence cleavage was detected. Protease digestion of mutants missing the C-terminal hydrophobic domain indicated that the C-terminus has stop transfer activity. A comparison of membrane insertion of the wild-type fusion protein to that of a mutant missing the second hydrophobic domain, the fusion sequence, indicated that the fusion domain has stop-transfer activity when synthesized in vitro. Furthermore, the fusion domain shows little signal sequence activity when positioned near the amino terminus of the fusion protein. The fusion protein has a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit. In order to determine the role that the conserved leucines have for the oligomeric structure and biological activity of the NDV fusion protein, the heptadic leucines at positions 481,488, and 495 were changed individually and in combination to an alanine residue. Whereas single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein although cell surface expression of the mutants and sedimentation in sucrose gradients was similar to that of the wild type. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain resulted in secretion of an oligomeric structure. These results indicate that the conserved leucines do not play a role in oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative substitutions of a serine-to-alanine (position 473), glutamic acid-to-lysine (position 482) or an asparagine-to-lysine (position 485), the fusogenic ability of the protein was not significantly disrupted. A phenylalanine residue is at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains which have a leucine residue in this position. To explore the role of this phenylalanine in the fusion activity of the protein, this residue was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. Whereas both the wild-type and the F117G proteins were proteolytically cleaved and F1 was detected, the leucine subsitution abolished cleavage. When co-expressed with the HN protein, the fusion protein with either a phenylalanine and glycine residue at position 117, but not a leucine, was shown to stimulate membrane fusion. However, incubation in trypsin activated the fusion activity of the F117L protein. Thus the presence of a leucine at position 117 of the precursor sequence blocks cleavage, but not fusion acitivity, and indicated that the phenylalanine at the amino terminus of the F1 subunit is not conserved for the fusion activity of the protein.

Drug resistance is the most important factor that influences the successful treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1), the causative organism of the acquired immunodeficiency syndrome (AIDS). Tremendous advances in our understanding of HIV and AIDS have led to the development of Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, to combat the virus. Though HAART has been successful in reducing AIDS-related morbidity and mortality, HIV rapidly evolves resistance leading to therapy failure. Thus, a better understanding of the mechanisms of resistance will lead to improved drugs and treatment regimens. Protease inhibitors (PIs) play an important role in anti-retroviral therapy. The development of resistance mutations within the active site of the protease greatly reduces its affinity for the protease inhibitors. Frequently, these mutations reduce catalytic efficiency of the protease leading to an overall reduction in viral fitness. In order to overcome this loss in fitness the virus evolves compensatory mutations within the protease cleavage sites that allow the protease to continue to recognize and cleave its substrates while lowering affinity for the PIs. Improved knowledge of this substrate co-evolution would help better understand how HIV-1 evolves resistance and thus, lead to improved therapeutic strategies. Sequence analyses and structural studies were performed to investigate co-evolution of HIV-1 protease and its cleavage sites. Though a few studies reported the co-evolution within Gag, including the protease cleavage sites, a more extensive study was lacking, especially as drug resistance was becoming increasingly severe. In Chapter II, a small set of viral sequences from infected individuals were analyzed for mutations within the Gag cleavage sites that co-occurred with primary drug resistance mutations within the protease. These studies revealed that mutations within the p1p6 cleavage site coevolved with the nelfinavir-resistant protease mutations. As a result of increasing number of infected individuals being treated with PIs leading to the accumulation of PI resistant protease mutations, and with increasing efforts at genotypic and phenotypic resistance testing, access to a larger database of resistance information has been made possible. Thus in Chapter III, over 39,000 sequences were analyzed for mutations within NC-p1, p1-6, Autoproteolysis, and PR-RT cleavage sites and several instances of substrate co-evolution were identified. Mutations in both the NC-p1 and the p1-p6 cleavage sites were associated with at least one, if not more, primary resistance mutations in the protease. Previous studies have demonstrated that mutations within the Gag cleavage sites enhance viral fitness and/or resistance when they occur in combination with primary drug resistance mutations within the protease. In Chapter III viral fitness in the presence and absence of cleavage site mutations in combination with primary drug resistant protease mutations was analyzed to investigate the impact of the observed co-evolution. These studies showed no significant changes in viral fitness. Additionally in Chapter III, the impact of these correlating mutations on phenotypic susceptibilities to various PIs was also analyzed. Phenotypic susceptibilities to various PIs were altered significantly when cleavage site mutations occurred in combination with primary protease mutations. In order to probe the underlying mechanisms for substrate co-evolution, in Chapter IV, X-ray crystallographic studies were performed to investigate structural changes in complexes of WT and D30N/N88D protease variants and the p1p6 peptide variants. Peptide variants corresponding to p1p6 cleavage site were designed, and included mutations observed in combination with the D30N/N88D protease mutation. Structural analyses of these complexes revealed several correlating changes in van der Waals contacts and hydrogen bonding as a result of the mutations. These changes in interactions suggest a mechanism for improving viral fitness as a result of co-evolution. This thesis research successfully identified several instance of co-evolution between primary drug resistant mutations in the protease and mutations within NC-p1 and p1p6 cleavage sites. Additionally, phenotypic susceptibilities to various PIs were significantly altered as a result of these correlated mutations. The structural studies also provided insights into the mechanism underlying substrate co-evolution. These data advance our understanding of substrate co-evolution and drug resistance, and will facilitate future studies to improve therapeutic strategies.

A common phenotype observed in most cancers is chromosomal instability. This includes both structural and numerical chromosomal aberrations, which can promote carcinogenesis. The fusion gene CBFB/MYH11 is created by the structural chromosomal inversion(16)(p13.1q22), resulting in the fusion protein CBFβ-SMMHC, which blocks differentiation in hematopoietic progenitor cells. This mutation alone, however, is not sufficient for transformation, and at least one additional cooperating mutation is necessary. The role of wildtype Cbfb in modulating the oncogenic function of the fusion protein Cbfβ-SMMHC in mice was examined. Transgenic mice expressing the fusion protein, but lacking a wild-type copy of Cbfb, were created to model the effects of these combined mutations. It was found that wild-type Cbfb is necessary for maintaining normal hematopoietic differentiation. Consequently, complete loss of wild-type Cbfb accelerates leukemogenesis in Cbfb/MYH11 mice compared to mice expressing both the fusion and wild-type proteins. While there is no evidence in human patient samples that loss of wild-type Cbfb expression cooperates with the fusion protein to cause transformation, it is apparent from these experiments that wild-type Cbfβ does play a role in maintaining genomic integrity in the presence of Cbfβ-SMMHC. Experiments have also shown that loss of Cbfb leads to accumulation of hematopoietic progenitor cells, which may acquire additional cooperating mutations. Not unlike CBFB/MYH11, the human papillomavirus (HPV) E6 and E7 proteins are not sufficient for cellular transformation. Instead, high risk HPV E7 causes numerical chromosomal aberrations, which can lead to accumulation of additional cooperating mutations. Expression of HPV-16 E7 and subsequent downregulation of the retinoblastoma protein (Rb) has been shown to induce polyploidy in human keratinocytes. Polyploidy predisposes cells to aneuploidy and can eventually lead to transformation in HPV positive cells. There are several possible mechanisms through which E7 may lead to polyploidization, including abrogation of the spindle assembly checkpoint, cleavage failure, abrogation of the postmitotic checkpoint, and re-replication. Rb-defective mouse and human cells were found to undergo normal mitosis and complete cytokinesis. Furthermore, DNA re-replication was not found to be a major mechanism to polyploidization in HPV-E7 cells upon microtubule disruption. Interestingly, upon prolonged mitotic arrest, cells were found to adapt to the spindle assembly checkpoint and halt in a G1-like state with 4C DNA content. This post-mitotic checkpoint is abrogated by E7-induced Rb-downregulation leading to S-phase induction and polyploidy. This dissertation explores two examples of the multi-step pathway in human cancers. While certain genes or genetic mutations are often characteristic of specific cancers, those mutations are often not sufficient for transformation. The genetic or chromosomal abnormalities that they produce often stimulate the additional mutations necessary for oncogenesis. The studies with Cbfb/MYH11 and HPV E7 further exemplify the significance of numerical and structural chromosomal aberrations in multi-step carcinogenesis.

Primate lentiviruses encode four “accessory proteins” including Vif, Vpu, Nef, and Vpr/ Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. The functions of Vpx/ Vpr are not well understood. This study presents evidence that the Vpx proteins of HIV-2/ SIVSMpromote HIV-1 infection by antagonizing an antiviral restriction in myeloid cells. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in transovercame the restriction to HIV-1 and SIV infection. Similarly, the cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx rendered macrophages permissive to MLV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. This study further demonstrates that this restriction prevents transduction of quiescent monocytes by HIV-1. Although terminally differentiated macrophages are partially permissive to HIV-1, quiescent monocytes, which are macrophage precursors, are highly refractory to lentiviral infection. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection, suggesting the resistance of quiescent monocytes to HIV-1 transduction is governed by a restriction factor. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Introduction of Vpx into monocytes by pre-infection also rendered quiescent monocytes permissive to HIV-1 infection. Infection of monocytes by HIV-1 either with or without Vpx did not have an effect on temporal expression of CD71. In addition, Vpx increased permissivity of CD71– and CD71+cells to HIV-1 infection with no apparent bias. These results confirm that Vpx directly renders undifferentiated monocytes permissive to HIV-1 transduction without inducing their differentiation. The introduction of Vpx did not significantly alter APOBEC3G complex distribution, suggesting a restriction other than APOBEC3G was responsible for the resistance of monocytes to HIV-1. Collectively our results indicate that macrophages and monocytes harbor a potent antiviral restriction that is counteracted by the Vpx protein. The relative ability of primate lentiviruses and gammaretroviruses to transduce non-dividing myeloid-cells is dependent upon their ability to neutralize this restriction.

Telomere length has been shown to be a critical determinant of T cell replicative capacity and in vivo persistence in humans. We evaluated telomere lengths in virus-specific T cells to understand how they may both shape and be changed by the maintenance of memory T cells during a subsequent virus re-infection or reactivation. We used longitudinal peripheral blood samples from healthy donors and samples from a long-term HCV clinical interferon therapy trial to test our hypotheses. To assess T cell telomere lengths, I developed novel modifications to the flow cytometry fluorescence in situ hybridization (flowFISH) assay. These flowFISH modifications were necessary to enable quantification of telomere length in activated, proliferating T cells. Adoption of a fixation-permeabilization protocol with RNA nuclease treatment prior to telomere probe hybridization were required to produce telomere length estimates that were consistent with a conventional telomere restriction fragment length Southern blot assay. We hypothesized that exposure to a non-recurring, acute virus infection would produce memory T cells with longer telomeres than those specific for recurring or reactivating virus infections. We used two acute viruses, vaccinia virus (VACV) and influenza A virus (IAV) and two latent-reactivating herpesviruses, cytomegalovirus (CMV) and varicella zoster virus (VZV) for these studies. Combining a proliferation assay with flowFISH, I found telomeres in VACV-specific CD4 + T cells were longer than those specific for the recurring exposure IAV; data which support my hypothesis. Counter to my hypothesis, CMV-specific CD4 + T cells had longer telomeres than IAV-specific CD4 + T cells. We assessed virus-specific CD4 + T cell telomere length in five donors over a period of 8-10 years which allowed us to develop a linear model of average virus-specific telomere length changes. These studies also found evidence of long telomere, virus-specific CD45RA + T cell populations whose depletion may precede an increased susceptibility to latent virus reactivation. I tested the hypothesis that type I interferon therapy would accelerate T cell telomere loss using PBMC samples from a cohort of chronic hepatitis C virus patients who either did or did not receive an extended course of treatment with interferon-alpha. Accelerated telomere losses occurred in naïve T cells in the interferon therapy group and were concentrated in the first half of 48 months of interferon therapy. Steady accumulation of CD57 + memory T cells in the control group, but not the therapy group, suggested that interferon also accelerated memory turnover. Based on our data, I present proposed models of memory T cell maintenance and impacts of T cell telomere length loss as we age.

Human papillomavirus infection is the cause of more than 99% of cervical cancer cases. The current vaccine is ineffective therapeutically; highlighting the need for continued papillomavirus research. One avenue that could be explored in this regard is the function of the papillomavirus E2 regulatory proteins. HPV E2 represses expression of the viral E6 and E7 oncoproteins. Reintroduction of E2 into cervical carcinoma cells results in growth arrest and cellular senescence. Understanding the mechanism of how E2 regulates the early promoter may be key to developing new therapeutic and prophylactic vaccines. Here, we describe regulation of E2 through acetylation and possibly through direct interaction with a novel cellular interacting protein, RINT1. Histone acetyltransferase (HAT) proteins have been demonstrated to interact with Bovine Papillomavirus (BPV) and Human Papillomavirus (HPV) E2 proteins as well as enhance E2 dependant transcription luciferase reporter plasmid containing E2 binding sites. We demonstrate that HATs p300, CBP, and pCAF are limiting for E2 dependant transcriptional activation and that each protein functions independently. We have also identified that BPV-1 E2 is a substrate for acetylation by p300. Mutants of E2 that cannot be acetylated on lysines 111 or 112, display abnormal transcriptional phenotypes. Cells deficient in p300 display similar transcriptional defects that are intensified by CBP depletion. We propose that acetylation of BPV-1 E2 is necessary for transcriptional activation. Acetylation generates a binding site through which a co-factor may interact via a bromodomain. Regulation of E2 dependent transcriptional activation through a post-transcriptional modification represents a novel method through which BPV-1 controls gene expression. We also present evidence for a direct interaction between BPV-1 E2 and the cellular factor RINT1. This interaction does not appear to be critical for transcriptional regulation; however, several other functional pathways are indicated by the cellular complexes in which RINT1 functions. Some of these, such as ER/Golgi vesicular transport and hTERT independent telomere maintenance, are pathways in which E2 has no known role. Further investigation into regulation and consequences of E2 acetylation and the biological significance of the interaction between E2 and RINT1 could prove important in understanding the complex role of E2 in papillomavirus infection.

Vpu has been shown to possess two distinct roles in the pathogenesis of HIV. First, Vpu has been shown to down-regulate the expression of CD4 molecules at the plasma membrane of infected cells by targeting CD4 molecules for degradation in the endoplasmic reticulum. Second, Vpu promotes viral egress in specific cell lines termed non-permissive cells by mechanism that remain relatively unclear. Therefore, experiments were conducted in order to identify cellular factors involved in the Vpu-dependent phenotype. Using full-length Vpu as bait in yeast two-hybrid experiments, several candidate cellular factors were identified. One protein, SNAPIN, was identified as a cellular factor putatively involved in the Vpu-dependent phenotype. Further experiments determined that not only do SNAPIN and Vpu interact, but that Vpu also leads to the degradation of SNAPIN by both proteasomal and lysosomal degradation pathways. Over-expression of SNAPIN in cell lines that do not normally require Vpu expression for viral production resulted in a Vpu-dependent phenotype. While over-expression of SNAPIN in otherwise permissive cell lines significantly reduced Vpu-deficient virus production, wild type levels remained relatively constant. Importantly, no defective viral structural protein production was observed; however, intracellular p24/p55 did not accumulate suggesting that in SNAPIN expressing cells, Gag is also targeted for degradation. In addition, the reduction of SNAPIN expression in non-permissive cell lines significantly increased viral titers in supernatants. Of particular interest, even in cells expressing Bst-2 (a previously identified cellular factor involved in the Vpu-phenotype), siRNA mediated knockdown of SNAPIN led to increased viral titers. In addition, the co-transfection of siRNAs targeting both SNAPIN and Bst-2 resulted in an additive effect, in which Vpu-deficient viral titers were nearly equivalent to wild-type titers. Surprisingly, siRNA-mediated knockdown of SNAPIN in Jurkat cells was sufficient to overcome any restriction in viral egress imposed by the deletion of Vpu. Conversely, siRNA targeting Bst-2 had little or no effect on viral titers in Jurkat cells regardless of whether it was transfected alone or in combination with siRNAs targeting SNAPIN. These experiments provide evidence of an alternate cellular restriction mechanism involved in viral egress that is countered by the HIV-1 accessory protein, Vpu. In addition, this research may provide further insight into the complex cellular networks involved in the trafficking of Gag through cellular endosomal pathways.

Around thirty years ago HIV-1 was identified, and from that point the known epidemic has grown to over 30 million infected individuals. Early on in the course of HIV-1 research, viruses were classified as either syncytia inducing, CXCR4-using, T-cell tropic or non-syncytia inducing, CCR5-using, macrophage tropic. Since that time, several groups have shown that this is an oversimplification. There is a great deal of diversity amongst CCR5-using HIV-1 variants. There remains a great deal to be discovered regarding HIV-1 CCR5-tropism and how this affects other aspects of HIV-1 infection. The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. In this thesis, a single amino acid determinant is described in the V1 loop that also modulates macrophage tropism. I identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection. In addition to determinants, this thesis seeks to evaluate the roles of macrophage tropic and non-macrophage tropic envelopes during the course of infection. Non-macrophage tropic virus predominates in immune tissue throughout infection, even in individuals suffering from HIV-associated dementia (HAD) who are known to carry many macrophage tropic viruses. There must be some advantage for these non-macrophage tropic viruses allowing them to persist in immune tissue throughout the disease. This thesis demonstrates that there is no advantage for these viruses to directly infect CD4+ T-cells, nor is there an advantage for them to be preferentially transmitted by dendritic cells to CD4+ T-cells. Given that transmitted/founder (T/F) viruses may preferentially interact with α4β7, and T/F viruses are non-macrophage tropic, I tested whether non-mac viruses could utilize α4β7 to their advantage. These experiments show that macrophage tropism does not play a role in gp120 interactions with α4β7. I evaluated whether there was a distinct disadvantage to macrophage tropic Envs, given their ability to infect dendritic cells and possibly stimulate the innate immune response. Using infected monocyte-derived dendritic cells (MDDCs), it was shown that mac-tropic Envs do not generate a significant immune response. These experiments demonstrate that there does not appear to be any advantage to non-macrophage tropic Envs, and that macrophage tropic Envs are able to infect CD4+ T-cells more efficiently, as well as DCs.

Gene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on CTD phosphorylation, but not on nascent RNA. Capping and methylation doesn't occur until nascent pre-mRNA become 19-22 nucleotides long. A second and novel pathway for recruiting and activating Mce1 involved direct physical interaction between the CTD, Tat and Mce1. Tat stimulated the guanylyltransferase and triphosphatase activities of Mce1, thereby enhancing the otherwise low efficiency of cotranscriptional capping of HIV mRNA. These findings imply that multiple mechanisms exist for coupling transcription elongation and mRNA processing at a checkpoint critical to HIV gene expression. To elucidate P-TEFb's function in human (HeLa) cells, RNA interference (RNAi) was used to degrade mRNA for hCycT1 or CDK9. Down-regulation of P-TEFb expression by RNAi can be achieved without causing major toxic or lethal effects and can control Tat transactivation and HIV replication in host cells. High-density oligonucleotide arrays were used to determine the effect of P-TEFb knockdown on global gene expression. Of 44,928 human genes analyzed, 25 were down-regulated and known or likely to be involved in cell proliferation and differentiation. These results provide new insight into P-TEFb function, its potent role in early embryonic development and strong evidence that P-TEFb is a new target for developing AIDS and cancer therapies. To fulfill the promise of RNAi for treating infectious and human genetic diseases, structural and functional mechanisms underlying RNAi in human cells were studied. The status of the 5' hydroxyl terminus of the antisense strand of short interfering RNA (siRNA) duplexes determined RNAi activity, while a 3' terminus block was tolerated in vivo. A perfect A-form helix in siRNA was not required for RNAi, but was required for antisense-target RNA duplexes. Strikingly, crosslinking siRNA duplexes with psoralen did not completely block RNAi, indicating that complete unwinding of the siRNA helix is not necessary for RNAi in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

Viral phenomenon in marketing refers to the rapid growth and adoption pattern of a product, akin to a biological virus. Viral phenomenon can be defined as “a word-of-mouth diffusion process wherein a message is actively forwarded from person to person, within and between multiple weakly linked personal networks, and is marked by a period of exponential growth in the number of people who are exposed to the message”. The success of Hotmail, a free web-based e-mail, and an Indian movie song ‘Kolavari Di’, are examples of viral phenomenon. Today, business commonly accepts mention of viral market penetration with reference to online videos, product, technology, information, an event or an idea. The need for attaining viral phenomenon is most critical in present times due to the dynamic market environment, the shrinking window of product lifecycle and multitude of competing products in the market. However, the measurement of viral success remains under explored in literature. The challenges and research gaps are: (i) The current studies are mostly case summaries without reliable metrics and lack empirical investigation. (ii) The scant empirical studies focus on a single measure of viral phenomenon, thereby do not capture the viral phenomenon in entirety. (iii) In the present time we are inundated with the term ‘viral phenomenon’ with no distinction between hype, popularity and what can be termed as a truly viral phenomenon. Moreover, the diversity of the occurrence of viral phenomenon across different mediums, for example product, online videos, an idea, further adds to the challenge of measuring the viral phenomenon. Researchers are yet to understand the viral phenomenon in its entirety and comprehensively. This thesis addresses these research gaps by defining the following research aims: 1) Research aim 1: Growth measurement of viral phenomenon – Development of a new viral phenomenon growth measurement methodology and to test and validate the proposed methodology for freely available online videos and purchased physical products. 2) Research aim 2: Viral phenomenon performance measures – Explore valid indicators to measure the viral phenomenon. 3) Research aim 3: Emotion measurement of viral phenomenon – Explores the emotion triggers that drive viral phenomenon thereby involves the development of a viral phenomenon emotion measurement methodology. This research makes three significant contributions to literature. (i) A new methodological framework is developed to comprehensively measure and characterize a viral phenomenon thereby eliminating the existing limitations of a single measure. (ii) This study is a first of its kind to empirically measure viral phenomenon of purchased physical products (i.e., non-online products purchased with monetary value). (iii) The methodology develops a viral emotion score to identify the viral emotions that trigger viral phenomenon.

ABSTRACT Learning through epiphanies, especially those not born out of dire crises, can be exciting, hugely satisfying, and life-validating ways to augment the significance of a lesson. In a moment of clarity, they have the ability to transform knowledge into wisdom, to provide a sense of interconnectedness within the grander scheme, and to contribute instant resolutions to persistent dilemmas seemingly irresolvable by less extraordinary means. This study involves a hermeneutic phenomenological investigation of epiphanies as they occur in lived experience. Its primary goal was to address the question, “What does it mean to have an epiphany?” Using data collected from 16 first-person, retroactive accounts of the experience, an analysis strategy using six spectra of contrasting dimensions was used to highlight the importance of the interplay between various characteristics of epiphanies. A significant finding of the study is that there is no such thing as an archetypical epiphany. Nevertheless, we can still recognize the phenomenon as a coherent entity mainly because there is agreement about many of its characteristics in the moment that it happens. Having an epiphany involves inductively rearranging patterns of deductively organized information with personally, and sometimes universally significant results. These may include dramatic shifts of perspectives, impulses that lead us to action on important matters, and occasionally whole-scale transformations of identity. Enhancing formal education settings to facilitate the experience can renew one’s passion for learning by making lessons more personally meaningful. In order to reap the benefits of the experience, we must hold space for the creative and introspective pursuits that allow them to happen. However, a challenge for facilitators of epiphanies involves learning how to direct the result of an epiphany to an intended outcome, such as a curricular goal.
Thesis (Master, Education) -- Queen's University, 2009-09-16 08:21:56.269

Many bulk properties of gases depend linearly on the gas density at lower densities, but as the density increases departures from linearity are observed. The density dependence of a bulk property Q may often be discussed systematically by expanding Q as a power series in l/Vm, to yield: Q=Aq+Bq/Vm+Cq/V2m+..., where Bq is known as the second virial coefficient of the property Q. Aq is the ideal gas value of Q, and Bq describes the contribution of molecular pair interactions to Q. Theories of Q may be regarded as having two main components, one describing how the presence of a neighbour of a given molecule can enhance or detract from its contribution to Q, and the other the molecular interaction energy which determines the average geometry of a pair encounter. The latter component is common to all theories, and the former requires detailed derivations for each specific bulk property Q. In this work we consider the second virial coefficients of five effects, namely the second pressure virial coefficient B(T), and also the second dielectric, refractivity, Kerr-effect and light-scattering virial coefficients, Be, Br, Bk, and Bp, respectively. Using a powerful computer algebraic manipulation package we have extended the existing dipole-induced-dipole (DID) theories of the second dielectric, refractivity and Kerr-effect virial coefficients to sufficiently high order to establish convergence in the treatment of both linear and non-linear gases. Together with the established linear theory of the second pressure virial coefficient, the extended theory of the second light- scattering virial coefficient developed by Couling and Graham, and their new non-linear theory of the second pressure and light-scattering virial coefficients, our new theories provide a comprehensive base from which to calculate numerical values for the various effects for comparison with experiment. We have collected as much experimental data of the various second virial coefficients as possible, for a wide range of gases. The ten gases chosen for detailed study comprise a selection of polar and non-polar, linear and non-linear gases: the linear polar gases fluoromethane, trifluoromethane, chloromethane and hydrogen chloride; the non-polar linear gases nitrogen, carbon dioxide and ethane; the non-linear polar gases sulphur dioxide and dimethyl ether; and the non-linear non-polar gas ethene. Using the best available measured or calculated molecular parameter data for these gases, together with the complete theories for the second virial coefficients, we have attempted to find unique sets of molecular parameters for each gas which explain all the available experimental data. In general, reliable measured or calculated molecular properties are regarded as fixed, and only the Lennard-Jones and shape parameters in the molecular interaction energy are treated as best-fit parameters within the constraints of being physically reasonable. Many of the apparent failures of second virial coefficient theories have been due to the lack of convergence in the series of terms evaluated. It is essential to work to sufficiently high orders in the polarizabilities and various multipole moments to ensure convergence for meaningful comparison with experiment. This often requires the manipulation of extremely long and complicated expressions, not possible by the manual methods of our recent past. The advent of computer manipulation packages and fast processors for numerical integration have now enabled calculation to high orders, where the degree of convergence can be sensibly followed. Our efforts to describe all of the effects for which data is available met with mixed success. For four of the gases, fluoromethane, chloromethane, dimethyl ether and ethene, a unique parameter set was found for each which described all of the available effects reasonably well. For the three gases, trifluoromethane, nitrogen and sulphur dioxide, one interaction parameter set explained all but one of the effects for which data was available to within experimental uncertainty. For trifluoromethane the parameter set which yielded good agreement for B(T), Be, and Bk could not explain the observed values of Br, while for nitrogen one parameter set produced reasonable agreement for all of the effects except Bp and a different set, which yielded good agreement for Bp, did not explain the remaining four effects as well as the first set. The parameter set which explained B(T), Bk and Bp very well for sulphur dioxide, yielded a value for Be, which was much larger than the experimental value, although of the correct sign and order of magnitude. Hydrogen chloride posed a special problem as data was only available for two of the effects, B(T) and Be. It was possible to find a set of interaction parameters in good agreement with the measured values of B(T), but the experimental data for Be was an order of magnitude larger than the largest calculated values. Since the remaining effects have not been measured for this gas it was not possible to test the theory more rigorously. For the remaining gases carbon dioxide and ethane, it was impossible, based on the existing measured values, to select a unique parameter set which explained all of the effects. In many of the cases where definite conclusions could not be drawn, it was not possible to decide whether the disagreement between theory and experiment was due to the large scatter and uncertainty of the experimental data or failure of the theory. However, there were very few instances of complete failure of the theory to explain experiment, and no one effect showed consistent disagreement, so that in general it may be said that the mechanisms of the second virial coefficients under study are reasonably well understood. It would require more precise measurements of the various effects, as well as more measured or calculated molecular property tensor components, such as the hyperpolarizability and the A- and C-tensors , to test the DID molecular interaction model more stringently.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.

Social phenomena have been studied extensively in small scales by social scientists. With the increasing popularity of Web 2.0 and online social networks/media, a large amount of data on social phenomena have become available. In this dissertation we study online social phenomena such as social influence in social networks in various contexts. This dissertation has two major components: 1. Identifying and characterizing online social phenomena 2. Leveraging online social phenomena for economic and commercial purposes. We begin the dissertation by developing multi-level revenue sharing schemes for viral marketing on social networks. Viral marketing leverages social influence among users of the social network. For our proposed models, we develop results on the computational complexity, individual rationality, and potential reach of employing the Shapley value as a revenue sharing scheme. Our results indicate that under the multi-level tree-based propagation model, the Shapley value is a promising scheme for revenue sharing, whereas under other models there are computational or incentive compatibility issues that remain open. We continue with another application of social influence: social advertising. Social advertising is a new paradigm that is utilized by online social networks. Social advertising is based in the premise that social influence can be leveraged to place ads more efficiently. The goal of our work is to understand how social ads can affect click-through rates in social networks. We propose a formal model for social ads in the context of display advertising. In our model, ads are shown to users one after the other. The probability of a user clicking an ad depends on the users who have clicked this ad so far. This information is presented to users as a social cue, thus the click probability is a function of this cue. We introduce the social display optimization problem: suppose an advertiser has a contract with a publisher for showing some number (say B) impressions of an ad. What strategy should the publisher use to show these ads so as to maximize the expected number of clicks? We show hardness results for this problem and in light of the general hardness results, we develop heuristic algorithms and compare them to natural baseline ones. We then study distributed content curation on the Web. In recent years readers have turned to the social web to consume content. In other words, they rely on their social network to curate content for them as opposed to the more traditional way of relying on news editors for this purpose -- this is an implicit consequence of social influence as well. We study how efficient this is for users with limited budgets of attention. We model distributed content curation as a reader-publisher game and show various results. Our results imply that in the complete information setting, when publishers maximize their utility selfishly, distributed content curation reaches an equilibrium which is efficient, that is, the social welfare is a constant factor of that under an optimal centralized curation. Next, we initiate the study of an exchange market problem without money that is a natural generalization of the well-studied kidney exchange problem. From the practical point of view, the problem is motivated by barter websites on the Internet, e.g., swap.com, and u-exchange.com. In this problem, the users of the social network wish to exchange items with each other. A mechanism specifies for each user a set of items that she gives away, and a set of items that she receives. Consider a set of agents where each agent has some items to offer, and wishes to receive some items from other agents. Each agent would like to receive as many items as possible from the items that she wishes, that is, her utility is equal to the number of items that she receives and wishes. However, she will have a large dis-utility if she gives away more items than what she receives, because she considers such a trade to be unfair. To ensure voluntary participation (also known as individual rationality), we require the mechanism to avoid this. We consider different variants of this problem: with and without a constraint on the length of the exchange cycles and show different results including their truthfulness and individual rationality. In the other main component of this thesis, we study and characterize two other social phenomena: 1. friends vs. the crowd and 2. altruism vs. reciprocity in social networks. More specifically, we study how a social network user's actions are influenced by her friends vs. the crowd's opinion. For example, in social rating websites where both ratings from friends and average ratings from everyone is available, we study how similar one's ratings are to each other. In the next part, we aim to analyze the motivations behind users' actions on online social media over an extended period of time. We look specifically at users' likes, comments and favorite markings on their friends' posts and photos. Most theories of why people exhibit prosocial behavior isolate two distinct motivations: Altruism and reciprocity. In our work, we focus on identifying the underlying motivations behind users' prosocial giving on social media. In particular, our goal is to identify if the motivation is altruism or reciprocity. For that purpose, we study two datasets of sequence of users' actions on social media: a dataset of wall posts by users of Facebook.com, and another dataset of favorite markings by users of Flickr.com. We study the sequence of users' actions in these datasets and provide several observations on patterns related to their prosocial giving behavior.