Prostate cancer (CaP) is the second leading cause of cancer related deaths in men in the United States. Currently, androgen depletion is an essential strategy for CaP combined with surgery, chemotherapy and radiation. Hormone independent cancer stem cells escaping conventional therapy present a major therapeutic challenge. The available treatment regimens for hormone resistant CaP are only palliative and marginally increase survival. Therefore, novel strategies to eradicate CaP including stem cells are imperative. Oncolytic virus (OV) therapy is a novel approach that overcomes the limitations posed by radiation and chemotherapy. Oncolytic virotherapy of cancer is based on the use of replication competent, tumor selective viruses with limited toxicity. Newcastle Disease Virus (NDV), an avian paramyxovirus, is a safe and promising OV successfully used in many clinical trials. NDV is inherently tumor selective and cytotoxic but replication restricted in normal cells. But, systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity and extracellular matrix. Overcoming these hurdles is paramount to realize the exceptional oncolytic efficacy of NDV. Therefore, we engineered the fusion (F) glycoprotein of NDV and generated a recombinant NDV (rNDV) cleavable exclusively by prostate specific antigen (PSA). The rNDV replicated efficiently and specifically only in prostate cancer (CaP) cells but failed to replicate in the absence of PSA. Further, PSA-cleavable rNDV caused specific lysis of androgen independent and dependent/responsive CaP cells with a mean effective concentration (EC50) ranging from 0.01 to 0.1 multiplicity of infection (MOI). PSA retargeted rNDV efficiently lysed three-dimensional prostaspheres, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating absence of pathogenicity to its natural host, chickens. Prostaspheres generated from DU-145 CaP cell line derived xenografts showed self-renewal, proliferative and clonogenic potential in vitro, and exhibited increased tumorigenicity in vivo. Embryonic stem and progenitor cell markers like Nanog, Nestin and CD44 were overexpressed in spheres as compared to the cell line suggesting prostaspheres comprise tumor-initiating cells from CaP. Xenograft and cell line derived prostaspheres were permissive for rNDV replication, when the fusion protein was activated by exogenous PSA. The EC50 against tumor initiating cells was 0.11-0.14 MOI, suggesting an excellent therapeutic margin for in vivo studies. PSA retargeting is likely to enhance the therapeutic index of rNDV owing to tumor restricted replication and enhanced fusogenicity. Our results suggest PSA retargeted rNDV selectively replicates and lyse PSA producing CaP cells including tumor-initiating cells and is a promising candidate for immediate Phase I/II clinical trials. / Ph. D.

Objetivou-se, neste trabalho, detectar o vírus da Influenza aviária, Paramyxovirus tipo 1 (doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae, respectivamente pelas técnicas de RT-PCR e PCR, em aves domésticas e aves em vida livre próximas às granjas avícolas nas cidades de Mogi das Cruzes e Louveira do Estado de São Paulo. As aves silvestres foram capturadas, anilhadas, submetidas à avaliação de estado geral e à coleta de suabes de orofaringe e cloaca. As aves de subsistência ou fundo de quintal seguiram o mesmo protocolo com a exceção do anilhamento, e tiveram amostras de sangue coletadas para a pesquisa de anticorpos contra o vírus da Doença de Newcastle, Mycoplasma gallisepticum e Mycoplasma synoviae pela técnica de ELISA indireto. Foram considerados os aspectos da biodiversidade entre as espécies silvestres capturadas e a biossegurança nas granjas. As aves silvestres apresentaram resultados negativos nesta pesquisa, no entanto, Mycoplasma gallisepticum e Mycoplasma synoviae foram detectados pela técnica da PCR nas aves de subsistência, assim como apresentaram títulos de anticorpos para os agentes acima citados e para o Paramyxovirus tipo I. Duas granjas não possuíam medidas de biosseguridade adequadas permitindo o contato de animais de vida livre com as aves de fundo de quintal e com as aves de produção, o que pode facilitar a disseminação de patógenos de interesse para a saúde pública e para a avicultura comercial. / The aim of this study is to detect avian influenza virus, Newcastle disease virus (Paramyxovirus I), Mycoplasma gallisepticum and Mycoplasma synoviae in backyard chicken and wildlife birds around commercial poultry farms using RT-PCR and PCR. The birds were captured with mist nets, identified with alluminium leg rings, subjected to the assessment of clinical conditions and samples were collected by oral and cloacal swabs. The same was done with backyard chicken without the identification with leg rings. Blood samples were collected from backyard chicken and tested for antibodies against Mycoplasma gallisepticum, Mycoplasma synoviae and Paramyxovirus I by indirect ELISA test. This study was conducted in Mogi das Cruzes and Louveira, São Paulo state, where the commercial poultry is considered an activity of great importance. The results were negative to wild birds, but we could detect Mycoplasma gallisepticum and Mycoplasma synoviae by PCR and antibodies titles for Mycoplasma gallisepticum, Mycoplasma synoviae and Newcastle disease in backyard chickens.Two farms didn´t have appropriate biosecurity measures, allowing intense contact with free-living birds, backyard chicken and poultry facilitating spread of pathogens with concern to human health and poultry farms.

Objetivou-se, neste trabalho, detectar o vírus da Influenza aviária, Paramyxovirus tipo 1 (doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae, respectivamente pelas técnicas de RT-PCR e PCR, em aves domésticas e aves em vida livre próximas às granjas avícolas nas cidades de Mogi das Cruzes e Louveira do Estado de São Paulo. As aves silvestres foram capturadas, anilhadas, submetidas à avaliação de estado geral e à coleta de suabes de orofaringe e cloaca. As aves de subsistência ou fundo de quintal seguiram o mesmo protocolo com a exceção do anilhamento, e tiveram amostras de sangue coletadas para a pesquisa de anticorpos contra o vírus da Doença de Newcastle, Mycoplasma gallisepticum e Mycoplasma synoviae pela técnica de ELISA indireto. Foram considerados os aspectos da biodiversidade entre as espécies silvestres capturadas e a biossegurança nas granjas. As aves silvestres apresentaram resultados negativos nesta pesquisa, no entanto, Mycoplasma gallisepticum e Mycoplasma synoviae foram detectados pela técnica da PCR nas aves de subsistência, assim como apresentaram títulos de anticorpos para os agentes acima citados e para o Paramyxovirus tipo I. Duas granjas não possuíam medidas de biosseguridade adequadas permitindo o contato de animais de vida livre com as aves de fundo de quintal e com as aves de produção, o que pode facilitar a disseminação de patógenos de interesse para a saúde pública e para a avicultura comercial. / The aim of this study is to detect avian influenza virus, Newcastle disease virus (Paramyxovirus I), Mycoplasma gallisepticum and Mycoplasma synoviae in backyard chicken and wildlife birds around commercial poultry farms using RT-PCR and PCR. The birds were captured with mist nets, identified with alluminium leg rings, subjected to the assessment of clinical conditions and samples were collected by oral and cloacal swabs. The same was done with backyard chicken without the identification with leg rings. Blood samples were collected from backyard chicken and tested for antibodies against Mycoplasma gallisepticum, Mycoplasma synoviae and Paramyxovirus I by indirect ELISA test. This study was conducted in Mogi das Cruzes and Louveira, São Paulo state, where the commercial poultry is considered an activity of great importance. The results were negative to wild birds, but we could detect Mycoplasma gallisepticum and Mycoplasma synoviae by PCR and antibodies titles for Mycoplasma gallisepticum, Mycoplasma synoviae and Newcastle disease in backyard chickens.Two farms didn´t have appropriate biosecurity measures, allowing intense contact with free-living birds, backyard chicken and poultry facilitating spread of pathogens with concern to human health and poultry farms.

<p>This thesis examines parental condition, how it is traded off against reproduction and how it is displayed in a secondary sexual trait. The studies were performed on nest-box breeding collared flycatchers Ficedula albicollis on the island of Gotland, in the Baltic Sea. Early breeding and high fitness were found to be associated with high levels of glycosylated haemoglobin possibly governed by migratory exertion and infectious disease. In order to test if immune function is expressed in secondary sexual traits and how it is traded off against reproductive effort a series of experiments were performed, in which birds were challenged with an antigen, via a vaccine containing neutralised paramyxovirus. The forehead patch of the male collared flycatcher serves as a badge of status and is under sexual selection. Good condition, as reflected in strong immune response and low levels of blood parasites was found to be associated with bigger patch size. Patch size was also found to vary in size within the same breeding season in a pattern predictable from immune response data. Immune response, in itself, was found to be costly in terms of reduced survival, confirming that trade-offs involving suppression of immune response may increase fitness. Mating effort was found to be traded off against immune function and moult. Experimental brood size manipulations revealed a trade-off females between number of offspring and immune function. Thus I suggest a set of parameters useful for condition estimation. I also show that immune response is costly and, second, that pathogen resistance probably plays an important role in the shaping of secondary sexual traits and life-history decisions.</p>

This thesis examines parental condition, how it is traded off against reproduction and how it is displayed in a secondary sexual trait. The studies were performed on nest-box breeding collared flycatchers Ficedula albicollis on the island of Gotland, in the Baltic Sea. Early breeding and high fitness were found to be associated with high levels of glycosylated haemoglobin possibly governed by migratory exertion and infectious disease. In order to test if immune function is expressed in secondary sexual traits and how it is traded off against reproductive effort a series of experiments were performed, in which birds were challenged with an antigen, via a vaccine containing neutralised paramyxovirus. The forehead patch of the male collared flycatcher serves as a badge of status and is under sexual selection. Good condition, as reflected in strong immune response and low levels of blood parasites was found to be associated with bigger patch size. Patch size was also found to vary in size within the same breeding season in a pattern predictable from immune response data. Immune response, in itself, was found to be costly in terms of reduced survival, confirming that trade-offs involving suppression of immune response may increase fitness. Mating effort was found to be traded off against immune function and moult. Experimental brood size manipulations revealed a trade-off females between number of offspring and immune function. Thus I suggest a set of parameters useful for condition estimation. I also show that immune response is costly and, second, that pathogen resistance probably plays an important role in the shaping of secondary sexual traits and life-history decisions.

Newcastle disease virus (NDV) is a member of the genus Avulavirus of the Paramyxoviridaefamily of enveloped negative-stranded RNA viruses. The virus causes respiratory, neurological, or enteric disease in many species of birds, resulting in significant losses to the poultry industry worldwide. Strains of the virus are classified into three pathotypes based on the severity of disease in chickens. Avirulent strains that produce mild or asymptomatic infections are termed lentogenic, whereas virulent strains are termed velogenic. Strains of intermediate virulence are termed mesogenic. The envelope of NDV virions contains two types of glycoproteins, the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. HN mediates three functions: 1) virus attachment to sialic acid-containing receptors; 2) neuraminidase activity that cleaves sialic acid from progeny virions to prevent self-aggregation; and, 3) complementation of the F protein in the promotion of fusion. Though it is widely accepted that cleavage of a fusion protein precursor is the primary determinant of NDV virulence, it is not the sole determinant. At least two other proteins, HN and the V protein, contribute to virulence. The V protein possesses interferon (IFN) antagonistic activity. The long-range goal of these studies is to understand the roles of HN and V in the differential virulence patterns exhibited by members of the NDV serotype. The first aim is to compare the IFN antagonistic activity of the V protein from a lentogenic and a mesogenic strain of the virus. The results of this study demonstrate that the V protein of the mesogenic strain Beaudette C (BC) exhibits greater IFN antagonistic activity than that of the lentogenic strain La Sota. Hence, the IFN antagonistic activities of the two V proteins correlate with their known virulence properties. Comparison of the C-terminal regions of La Sota and BC V proteins revealed four amino acid differences. The results demonstrate that the IFN antagonistic activity of La Sota V increases when any one of these residues is mutated to the corresponding residue in BC V. Conversely, the IFN antagonistic activity of BC V decreases when any one of these four residues is mutated to the corresponding residue in La Sota V. However, no single residue accounts for the difference in IFN antagonistic activity between the two V proteins. Also, analysis of La Sota V and BC V proteins with multiple mutations in these positions revealed that the four residues are collectively responsible for the difference in the IFN antagonistic activity of the two V proteins. Finally, characterization of chimeric La Sota/BC V proteins showed that the N-terminal region also contributes to the IFN antagonistic activity of V. Contrary to an earlier report, results described here demonstrate that the NDV V protein does not target STAT1 for degradation. However, both La Sota and BC V proteins target interferon regulatory factor (IRF)-7 for degradation and promote the conversion of full-length IRF-7 to a lower molecular weight form (IRF-7*). This is the first demonstration that IRF-7 is targeted by a paramyxovirus V protein. The amount of IRF-7* decreases in a dose-dependent manner in the presence of a proteasome inhibitor, suggesting that IRF-7* is a degradation product of IRF-7. Furthermore, the BC V protein promotes complete conversion of IRF-7 to IRF7*, whereas the La Sota V protein does so less efficiently. Again, this is consistent with the difference in IFN antagonistic activity of the two V proteins, and in turn, with their virulence. The second aim is to characterize an HN-specific monoclonal antibody called AVS-I. A previous study suggested that AVS-I recognizes an epitope that is conserved in lentogenic strains and raises the possibility that this epitope may colocalize with a determinant of virulence in HN. To further characterize antibody AVS-I and the epitope it recognizes, we (i) determined its specificity for several additional strains of the virus, (ii) mapped its binding to HN in competition with our own antibodies, (iii) determined its functional inhibition profile, and (iv) isolated and sequenced an AVS-I escape mutant. The results demonstrate that AVS-I binds to a conformational epitope at the carboxy terminus of HN. This suggests that this region of HN may define a determinant of virulence. However, it was also shown that AVS-I, which was previously thought to be specific for avirulent strains of NDV, actually recognizes individual mesogenic and velogenic strains. In conclusion, the data presented in this dissertation contributes to a greater understanding of the molecular basis for NDV virulence and may aid in development of antiviral strategies and generation of recombinant NDVs suitable for use in cancer and gene therapy.

It is shown here that the ability of B lymphocytes to act as supportive host cells for virus infections requires they be activated from the resting Gostage of the cell cycle. I have used a series of activation regimens, which allow B cells to progress to different stages in their activation/differentiation pathway toward antibody secretion, in order to evaluate the extent of activation required to support vesicular stomatitis or Newcastle disease virus infections. At least three distinct phases during B cell activation which affected VSV infection were defined. Freshly isolated resting murine splenic B cells in the Go phase of the cell cycle do not support VSV, assessed by protein synthesis, infectious center formation, and PFU production. Small B cells cultured for 48 hours without stimulation still do not support VSV. B cells stimulated with the lymphokines found in Con A activated supernatants from splenic T cells or cloned T cell lines transited into the G1 phase of the cell cycle but remain refractory to VSV. These VSV non-supportive B cell populations do take up virus particles and transcribe viral mRNAs which can be translated in vitro, suggesting a translational block to VSV. B cells stimulated into the S phase of the cell cycle with anti-immunoglobulin synthesize VSV proteins and increased numbers of infectious centers, but only low level PFU synthesis (center) is observed. Co-stimulation with anti-Ig and lymphokines, which supports differentiation to antibody secretion, enhanced PFU synthesis without further increasing the number of infected B cells. LPS, which activates B cells directly to antibody secretion by a pathway different from anti-Ig, induced infectious centers, and PFUs at levels comparable to those seen when stably transformed permissive cell lines are infected. Co-stimulation of LPS activated B cells with the same lymphokine populations that enhance PFU production when anti-Ig is used as a stimulator suppresses PFU production completely, suggesting that anti-Ig and LPS activated B cells are differentially responsive to lymphokines. NDV infection of murine B cells differed markedly from VSV infection, as all B cell populations examined gave a similar response pattern. NDV viral proteins were synthesized by B cells in each of the activation states previously described, even freshly isolated B cells. Infectious center formation increased up to 5-fold over the levels observed with unstimulated B cells after anti-Ig or LPS activation. However, PFU synthesis was low (center) for all B cell populations. These results suggest that these two similar viruses may be dependent on different host cell factors and that these factors are induced for VSV but not NDV by the B cell activators employed here or that the process of infection of B cell by these two viruses induces different cellular responses.

M.A. / The institutionalisation and rehabilitation of juvenile delinquents has always been a contentious issue, generating much research and differences in opinions. What to do with youngsters who are too young to be criminals and too violent to be youth, remains a complex dilemma in a society where the incidence of juvenile delinquency is increasing by the day. The South African phenomenon of a marginalised generation who readily takes part in criminal behaviour, necessitated the development of a co-ordinated strategy involving formal and informal support groups in the training and development of this group. The establishment of e ,e first Youth Development Centre in Newcastle, named Ekuseni, was initiat ,* by President Nelson Mandela, in response to his concern that the conditions in South African jails are not conducive to transforming and developing young prisoners. The Ekuseni project was aimed at providing young convicted persons with appropriate life skills, education and training, to enable them to pa cipate fully in society. The aim of this study was to develop a psycho-educational programme to facilitate leadership competencies in young prisoners. The leadership programme constitutes one of the development programmes in the holistic rehabilitation model, developed specifically for the Ekuseni project by the Rand Afrikaans University. The leadership programme is aimed at developing various leadership competencies, grounded in leadership competency theory. The competencies included in this study were more specifically based on the research and the development of a unique leadership model for the South African organisational context by Charlton (1993). These concepts were adapted and integrated with theory on juvenile delinquency to develop a leadership program= suitable for South African youth in prison. The first step in the programme was to help students to create a vision for themselves and for the Ekuseni Youth Development Centre, and to take responsibility for attaining that vision. This included a shift from an external to an internal locus of control. Through learned communication skills, e competency to communicate this vision to other prisoners, to enlist them in dedicated action towards a constructive future, was facilitated. The development of conflict management skills as an essential competency for leaders in a youth prison, were facilitated in order for leaders to constructively resolve conflict between prisoners and staff, as well as between prisoners themselves. This is especially necessary in conflict between youth gangs in prison. Trust, earned by leaders through reliable and consistent behaviour is a fourth competency facilitated through IP is programme. Students were taught the art of interpersonal trust, which in turn enabled them to help others and empower themselves. The evaluation of the effectiveness of this programme did not fall nV in the parameters of this study. It is therefore recommended that this study be evaluated in future, before it is implemented in other youth prisons in South Africa.

“Circulating Knowledges: Literature and the Idea of the Library in Renaissance England” pairs literary texts and libraries to illustrate how literary creation and library building in England from 1500 to 1700 were deeply invested in one another. The history of English Renaissance libraries has generally been analyzed from the viewpoints of religious history and historiography, seen by scholars as a story of Protestant librarians attempting to preserve (or invent) a history of Protestant England. Many literary critics —citing Thomas Bodley’s notorious distaste for “stage plaies”—have typically reduced institutional libraries to elitist boogeymen hostile to popular or vernacular literature. Revising these narratives, this dissertation brings together a large corpus, including works by Thomas More, John Lyly, Edmund Spenser, Robert Greene, Christopher Marlowe, Francis Bacon, and Margaret Cavendish, to illustrate how literary depictions of England’s fledgling libraries shaped their creation and development, while the practices of these inchoate libraries in turn influenced literary texts. “Circulating Knowledges” advances its argument on several fronts. First, I show that developments (or a perceived lack of development) in library organization, access, and use appeared in literary texts, which often depicted literary libraries in response to these developments. Second, I home in on moments when literary texts that seem not at all interested in libraries become unexpectedly fruitful texts through which to develop literary thinking about libraries. In the process of excavating this literary interest in libraries, I demonstrate that Renaissance literature concerns itself not only with depicting, commenting on, or objecting to the developments in library creation happening during the period, but also in imagining alternative possibilities for how libraries might function, conceptions of a library that often outstripped what was materially possible in the period: these conceptions I term “the idea of the library.” In detailing literature’s preoccupation with developments in Renaissance library systems, I offer new perspectives on the period’s literary attitudes toward the creation, transmission, and protection of knowledge, all questions which the building—or imagining—of a library brings to the forefront.

The first step of viral infection requires the binding of the viral attachment protein to cell surface receptors. Following binding, viruses penetrate the cellular membrane to deliver their genome into the host cell. For enveloped viruses, which have a lipid bilayer that surrounds their nucleocapsids, entry into the host cell requires the fusion of viral and cellular membranes. This process is mediated by viral glycoproteins located on the surface of the virus. For many enveloped viruses, such as influenza, Ebola, and human immunodeficiency virus, the fusion protein is responsible for mediating both attachment to cellular receptors and membrane fusion. However, paramyxoviruses are unique among fusion promoting viruses because their receptor binding and fusion activities reside on two separate proteins. This unique distribution of functions necessitates a mechanism by which the two proteins can transmit the juxtaposition of the viral and host cell membranes, mediated by the attachment protein (HN/H), into membrane fusion, mediated by the fusion (F) protein. This mechanism allows for paramyxoviruses to gain entry into and spread between cells, and therefore, is an important aspect of virus infection and disease progression. Despite the conservation of receptor binding activity among members of the Paramyxovirinaesubfamily, for most of these viruses, including Newcastle disease virus (NDV), heterologous HN proteins cannot complement F in the promotion of fusion; both the HN and F proteins must originate from the same virus. This is consistent with the existence of a virus-specific interaction between the two glycoproteins. Thus, one or more domains on the HN and F proteins is thought to mediate a specific interaction between them that is an integral part of the fusion process. Therefore, the primary focus of this thesis is the identification of the site(s) on HN that directly contacts F in the HN-F interaction. The ectodomain of the HN protein consists of a stalk and a terminal globular head. Analysis of the fusion activity of chimeric paramyxovirus HN proteins indicates that the stalk region of HN determines its F protein specificity. The first goal of this research was to address the question of whether the stalk not only determines F-specificity, but does so by directly mediating the interaction with F. To establish a correlation between the amount of fusion and the extent of the HN-F interaction, a specific and quantitative co-immunoprecipitation assay was used that detects the HN-F complex at the cell surface. As an initial probe of the role of the HN stalk in mediating the interaction with F, N-glycans were individually added at several positions in the region. N-glycan addition at positions 69 and 77 in the stalk specifically and completely block both fusion and the HN-F interaction without affecting either HN structure or its other activities. However, though they also prevent fusion, N-glycans added at other positions in the stalk also modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein as indicated by sucrose gradient sedimentation analyses. These additional N-glycans likely indirectly affect fusion, perhaps by interfering with changes in the conformation of HN that link receptor binding to the fusion activation of F. To address the issue of whether N-glycan addition at any position in HN would abolish fusion, an N-glycan was added in another region at the base of the globular head of HN (residues 124-152), which was previously predicted by a peptide-based analysis to mediate the interaction with F. HN carrying this additional N-glycan exhibits significant fusion promoting activity, arguing against this site being part of the F-interactive domain in HN. These data support the idea that the F-interactive site on HN is defined by the stalk region of the protein. Site-directed mutagenesis was used to begin to explore the role of individual residues in the stalk in the interaction with F. The characteristics of the F-interactive domain in the stalk of HN are that it is a conserved motif with enough sequence heterogeneity to account for the specificity of the interaction. One such region that meets these requirements is the intervening region (IR) (residues 89-95); a non-helical domain situated between two conserved heptad repeats. Several amino acid substitutions for a completely conserved proline residue in this region impair not only fusion and the HN-F interaction, but also decrease neuraminidase activity in the globular domain and alter the structure of the protein, suggesting that the substitutions indirectly affect the HN-F interaction. Substitutions for L94 also interfere with fusion, but have no significant effect on any other HN function or its structure. Amino acid substitutions at two other positions in the IR (A89 and L90) also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the IR is critical to the role of HN in the promotion of fusion and are consistent with its direct involvement in the interaction with the homologous F protein. These are the first point mutations in the HN protein for which a correlation has been demonstrated between the extent of the HN-F interaction and the amount of fusion. This argues strongly that the co-IP assay is an accurate reflection of the HN-F interaction. The second goal of this research was to address the HN-F interaction from the perspective of the F protein by investigating the relationship between receptor binding, the HN-F interaction, and fusion using a highly fusogenic form of the F protein. It has previously been shown that an L289A substitution in NDV F eliminates the requirement for HN in the promotion of fusion and enhances HN-dependent fusion above wild-type (wt) levels. Here, it was shown that the HN-independent fusion exhibited by L289A-F in Cos-7 cells cannot be duplicated in BHK cells. However, when L289A-F is co-expressed with wt HN, enhanced fusion above wt levels is observed in BHK cells. Additionally, when L289A-F is co-expressed with IR-mutated HN proteins previously shown to promote low levels of fusion with wt F, a 2.5-fold increase in fusion was observed. However, similar to wt F, an interaction between L289A-F and the IR-mutated HN proteins was not detected. These results imply that the attachment function of HN, as well as the conformational change in L289A-F, are necessary for the enhanced level of fusion exhibited by HN proteins co-expressed with L289A-F. Indeed, two MAbs detected a conformational difference between L289A-F and the wt F protein. These findings support the idea that the L289A substitution converts F to a form that is less dependent on an interaction with HN for conversion to the fusion-active form. The last goal of this research was to address the cellular site of the HN-F interaction, still a controversial issue based on conflicting data from studies of different paramyxoviruses, using various approaches. This is a particular point of interest, as it speaks to the mechanism by which the HN-F interaction regulates fusion. Thus, NDV HN and F were successfully retained intracellularly with a multiple arginine or KK motif, respectively. The results of Endoglycosidase H resistance and F cleavage studies indicate that the mutated proteins, HN-ER and F-ER, are retained in a compartment prior to the medial-Golgi apparatus and that they are unable to interact with a high enough affinity to co-retain or even cause reduced transport of their wt partner glycoproteins. This is consistent with the HN-F interaction occurring at the cell surface, possibly triggered by receptor binding. In conclusion, this thesis presents evidence to argue that the IR in the stalk of the NDV HN protein directly mediates the interaction with the F protein that is necessary for fusion. Overall, the data presented in this thesis extend the current knowledge of the mechanism by which the paramyxovirus attachment protein can trigger the F protein to initiate membrane fusion. A clear understanding of this process has the potential to identify new anti-viral strategies, such as small molecule inhibitors, aimed at controlling paramyxovirus infection by interfering with early steps in the virus infection cycle.