<p>Corruption is a serious problem which has many negative impacts on sustainable economic development globally. The clandestine nature of corruption makes it difficult to detect. Hence, efforts to combat corruption successfully demand comprehensive anti-corruption legislation, strong powers, as well as special investigative techniques and strategies. An effective anti-corruption regime requires a comprehensive anti-corruption legal framework which not only punishes all forms of corruption but also capacitates anti-corruption institutions. A strong anti-corruption agency is a&nbsp / crucial requirement and a necessary part of a country&rsquo / s anti-corruption strategy. The failure or the success of an anti-corruption agency depends on a variety of factors, such as powers and means to detect, investigate and prosecute corruption and related offences. The lack of trained staff, as well as the lack of adequate material resources, also affects the effectiveness of an anti-corruption agency. The anti-corruption agencies covered by this research are not empowered or resourced sufficiently, which may result in their ineffectiveness. This paper provides a set of recommendations in respect of the powers and strategies needed for a successful anti-corruption agency.</p>

<p>Corruption is a serious problem which has many negative impacts on sustainable economic development globally. The clandestine nature of corruption makes it difficult to detect. Hence, efforts to combat corruption successfully demand comprehensive anti-corruption legislation, strong powers, as well as special investigative techniques and strategies. An effective anti-corruption regime requires a comprehensive anti-corruption legal framework which not only punishes all forms of corruption but also capacitates anti-corruption institutions. A strong anti-corruption agency is a&nbsp / crucial requirement and a necessary part of a country&rsquo / s anti-corruption strategy. The failure or the success of an anti-corruption agency depends on a variety of factors, such as powers and means to detect, investigate and prosecute corruption and related offences. The lack of trained staff, as well as the lack of adequate material resources, also affects the effectiveness of an anti-corruption agency. The anti-corruption agencies covered by this research are not empowered or resourced sufficiently, which may result in their ineffectiveness. This paper provides a set of recommendations in respect of the powers and strategies needed for a successful anti-corruption agency.</p>

Multiple Sclerosis (MS) is a neurological autoimmune disease characterized by demyelination of central nervous system tissue and one way this is presented is in the demyelination of the retinal nerve, causing vision disturbance and loss (Munger et al., 2006). The thinning of the retinal nerve fiber layer (RNFL) can be measured and visualized using a noninvasive technique called Optical Coherence Tomography (OCT), which is also used to measure relative MS severity (Petzold et al., 2010). One environmental factor that has been found to have a relationship with MS is vitamin D; research findings suggest that sufficient levels of vitamin D may reduce the risk of developing MS, decrease MS severity, and may slow its progression (Ascherio et al., 2010; Munger et al., 2006; Muris et al., 2013). The mechanism by which vitamin D affects certain symptoms requires deeper investigation. This research examines the relationship between serum concentrations of 25-hydroxyvitamin D and retinal nerve fiber layer thicknesses in patients with MS. It was hypothesized that patients with sufficient vitamin D levels would have less demyelination of the retinal nerve caused by MS, and therefore would have a thicker RNFL in both eyes based on the proposed immunomodulatory role of vitamin D found in other studies. Blood samples were assayed to measure the concentration of 25-hydroxyvitamin D and OCT was used to measure RNFL thicknesses in patients with MS at the Harbor-UCLA Medical Center Neurology Clinic. Patients with sufficient levels of 25-hydroxyvitamin D had a greater mean global RNFL thickness in both eyes than in patients with insufficient levels of 25-hydroxyvitamin D; however the differences were not significant. Further research is necessary in order to determine whether or not there is a correlation between vitamin D and RNFL thickness and what role vitamin D plays in MS presentation.

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed a superfluous choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layerspecific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway.

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.

Virus-specific cellular immune responses have been shown to be important in the control of viral replication in several animal and human virus models. Cells of both the CD8+ and CD4+T cell lineages have been shown to play protective roles during viral infections by exerting effector functions that can kill infected host cells or inhibit the production and spread of infectious virions. The continued spread of HIV-1 infection throughout the world, as well as the lack of a prophylactic HIV-1 vaccine have generated much interest in HIV-specific cellular immune responses. Recent technical advances have yielded a tremendous increase in our understanding of HIV-1-specific immunity, as well as HIV-1 replication dynamics and host cell factors that shape the course of acute and chronic infection. Unfortunately, due to small sample volumes and technological limitations, the study of HIV-1-specific T cell immunity in infants and children has been difficult. An improved understanding of the timing, specificity, and intensity of pediatric HIV-specific T cell responses would contribute to the development of a HIV-1 vaccine for use in regions of the developing world without access to antiretroviral therapeutics. In the small number of published studies investigating pediatric HIV-specific immunity, T cell responses were uncommonly detected in infants. It remains unclear, however, whether the lack of HIV-specific T cells is an accurate reflection of the in vivoimmune state in vertically-infected infants, or rather is a consequence of reagents and assays ill-suited to the detection of low-level and/or diverse T cell responses in pediatric subjects. In the present dissertation, several methodologies were used to investigate HIV-specific T cell responses in vertically-infected infants and children. HIV-specific CD8+ T cell responses were infrequently detected in a cohort of young infants, but are commonly detected in older infants and children. Interestingly, CMV-specific CD8+ T cell responses were detected in several young infants that lacked HIV-specific responses, suggesting a specific defect in the ability of some infants to generate HIV-specific CD8+ T cell responses. Further experiments characterizing detectable HIV-1-specific CD8+ T cell responses found that the HIV-1 accessory proteins may be important targets of the immune response during early vertical infection. The role of HLA class I genotype and viral sequence are also explored in a pair of vertically-infected twins with discordant CD8+T cell responses. Finally, viral isolates from an infant with a marked shift in gag-specific epitope usage during infancy are analyzed for the presence of escape mutations. Gag-specific CD4+ T cell responses were commonly detected in a large cohort of vertically-infected children. A linear relationship between HIV-1 replication and the presence and intensity of HIV-specific CD4+ T cell responses was found, but ongoing HIV-1 replication appeared to blunt CD4+T cell proliferation. The data presented in this dissertation describe pediatric T cell immune responses and how they relate to HIV-1 replication. This information may be useful to the design of a prophylactic or therapeutic HIV-1 vaccine for vertically-infected infants and children.

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.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus which causes acute infectious mononucleosis and is etiologically associated with malignant lymphoproliferative disorders including Burkitt's lymphoma, nasopharyngeal carcinoma, B-cell lymphomas in immunocompromised hosts, Hodgkin's disease, T cell lymphomas, and smooth muscle tumors in allograft recipients. The medical significance of EBV is underscored by its potent growth transforming effects on human B-lymphocytes in-vitro and the potentially oncogenic consequences of infection in-vivo. The majority of EBV-associated malignancies occur in the setting of chronic infection and strong virus-specific humoral immunity, suggesting that cellular immunity is primarily responsible for preventing the outgrowth of EBV-transformed B cells in-vivo. Similarly, primary EBV infection in adolescents and adults stimulates an intense cytotoxic-T-lymphocyte (CTL) response which coincides with a marked reduction in the number of infected B cells in the peripheral blood. Evidence of previous EBV infection can be confirmed by the presence of EBV-specific, HLA-restricted memory T cells in the peripheral blood which inhibit the outgrowth of newly EBV-transformed B cells and efficiently lyse established autologous B-lymphoblastoid cell lines. Worldwide, EBV is responsible for substantial morbidity, comparable to measles, mumps and hepatitis virus, for which vaccines exists. Accordingly, the potential public health impact of an EBV vaccine has reinforced our efforts to identify the immunodominant virus-encoded T-cell epitopes which stimulate naive CTL effectors during acute infection and maintain memory CTL surveillance during convalescence. The EBV-encoded antigens against which the memory CTL response is directed have been partially defined, and include most of the EBV latent proteins (EBNA-2, 3a, 3b, 3c, LP, and LMP-l, 2a, 2b) consistently expressed by in-vitro EBV-transformed B lymphocytes (type-III latency). Importantly, all EBV-associated malignancies express EBNA-1, and as yet no EBNA-1-specific memory CTL have been convincingly demonstrated. Additionally, many EBV-specific CTL lines and clones have been described which do not recognize any of the known latent proteins or other EBV protein antigens tested thus far. Thus while much is known about CTL-mediated immunity against EBV, our knowledge of EBV-derived CTL epitopes remains incomplete. In contrast to the EBV-specific memory CTL response, very little is known about the source of viral epitopes recognized during the primary CTL response to EBV. In this regard, acute infectious mononucleosis represents an ideal model system to study virus-specific, cell-mediated immunity. Acute IM is a self-limited illness characterized by the appearance of "atypical" lymphocytes (CD3+/CD8+/HLADR+), including both virus-specific and alloreactive CTL, which undoubtedly contribute to virus elimination and provide CTL precursors for life-long immunity to EBV. Like other herpesvirus, EBV can undergo either lytic or latent cycle replication. During primary EBV infection many lytic cycle genes are expressed which are likely responsible for stimulating the intense cellular immune response associated with acute infectious mononucleosis. During convalescence a minor population of circulating B cells remain latently infected, harbor multiple EBV episomes, and express only EBNA-1 and possibly LMP-2a (type-I latency). Thus, latency type-I infected B cells in-vivo express a much more restricted spectrum of latent proteins and are therefore not subject to elimination by the same virus-specific CTL as are type-III EBV latently infected cells. Accordingly, many mechanisms have been proposed to explain EBV persistence including; restricted expression of EBV latent genes, reduced levels of cellular adhesion molecules, downregulation of MHC class-I molecules, absence of EBNA-1 T-cell-epitopes, and most recently, EBNA-1-mediated inhibition of antigen processing. While these mechanisms may contribute to ineffective T cell surveillance against latency type-I EBV- infected cells, B cells expressing the full spectrum of latent proteins (type-III) also exist transiently in vivoand maintain detectable humoral and CTL responses to most latent proteins. Our first goal was to identify the virus-encoded immunodominant antigens recognized by in-vivoactivated MHC class-I restricted CTL isolated from college students experiencing primary EBV infection, manifested as acute IM. Following a prodromal period of several weeks, newly EBV infected patients present with signs and symptoms of acute IM, including elevated numbers of activated CD8+ T cells in their peripheral blood, many of which, like memory CTL, are EBV-specific and HLA-restricted. In order to address the issue of EBV persistence and the immune control of EBV-induced lymphoproliferation, we also studied the long-term EBV-specific memory CTL response in these same individuals. Blood from acute IM patients and healthy EBV seropositive donors served as a source of peripheral blood lymphocytes to generate bulk CTL cultures and autologous target cells. The infecting strain of EBV was determined for each patient by DNA-PCR amplification of virus from saliva. Lymphocytes were isolated from whole blood by Ficoll-Paque density centrifugation and T- and B-cell enriched populations were obtained by AET-sheep red cell rosette selection. Autologous B cell blasts served as a source of target cells and recombinant vaccinia virus constructs were used to introduce individual EBV latent genes into target cells. Expression of individual EBV genes in target cells was confirmed by both western blot and immunofluorescence. Primary CTL responses to EBV were evaluated in standard 5lCr release assays using freshly isolated, T-cell enriched PBL from acute IM patients as effector cells. EBV-specific memory CTL responses were evaluated with bulk CTL culture generated by in-vitro restimulation with autologous B-LCLs. FACS analyses were routinely performed on bulk cultures of effector CTL populations in order to more clearly characterize their phenotype. Lastly, monoclonal antibody blocking studies and cold target competition assays were performed in order to accurately identify the viral antigen and MHC components responsible for target cell recognition. Our results based upon evaluation of 35 acute IM patients and 32 convalescent patients demonstrate that the virus-specific primary CTL response is broadly directed against the full spectrum of latent proteins, including EBNA1 and the viral coat glycoprotein gp350, while the memoryCTL response, which essentially lacks EBNA1 reactivity, is directed primarily against the EBNA 3 family of proteins (3A, 3B, 3C). Importantly, the immunodominant response by both primary and memory CTL was directed against the EBNA3 proteins. CTL from 7 of the 35 acute IM patients evaluated recognized EBNA1 expressing targets, and in 4 of these 7 patients, EBNA1 was an immunodominant antigen. Similarly, CTL from 7 of 35 acute IM patients recognized gp350 transfected targets, while no gp350-specific memory CTL responses were observed. While the phenotype of in-vivo primed CTL effectors were CD8+/HLA-DR+/CD11b+, the major subpopulation of memory CTL were CD8+/HLA-DR+/CD11b-. The CD11b "memory marker" reached peaked levels on the first sample day for all patients and gradually declined to baseline levels over a period of several months. In contrast, the CD11b marker was quickly shed from in vitropropogated CTL, over a period of 5-10 days. Target cell lysis by in-vivoactivated CTL was almost completely blocked by antibody directed againt [against] class-I molecules (BBM.1), whereas the effect of blocking target cell lysis by anti-CD8 mAb varied between 40-75%. These findings are consistent with an absolute need for class-I restricted antigen presentation, and imply that CD8 was variably required, likely for the lower affinity TCR/ Ag combinations. Cell lysis mediated by in-vitro-restimulated memory CTL was also largely inhibited by anti-class-I mAb, while anti-CD8 mAb was only mild/moderately effective in blocking target cell lysis, in keeping with the concept that memory CTL bear higher avidity TCR which can recognize antigen independent of CD8. Our detection of only one EBNA1-specific memory CTL response among the 32 patients tested supports the theory that latently infected B cells in-vivo, expressing only EBNA1, escape CTL recogition and thus might serve as a reservoir for viral persistence and/or reactivation. The rare ability to detect an EBNA1-specific memory CTL responses remains a relatively unexplained phenomenon and may involve a number of tolerizing mechanisms including the induction of anergy by presentation of EBNA-1 in the absence of costimulation, clonal deletion of low affinity T cells, the absence of dominant T cell epitopes within EBNA1 or a result of the recently described inhibiting properties of EBNA-1 on antigen processing and presentation. Alternatively, the absence of detectable EBNA1-specific memory CTL may be the result of insufficient or inappropriate restimulation of memory CTL in vitro. We addressed this possibility by attempting to selectively restimulate and expand EBNA1-specific CTL from acute IM patients by using EBNA1 expressing B cells blasts as a stimulus. Effector cells generated in this manner killed target cells in an MHC class-I restricted manner but were specific for an unspecified vaccinia antigen. Interestingly, the phenotype of the effector cells was predominantly CD3+/CD4-/CD8-/γδ T cells. In summary, our findings suggest that a multitude of previously unrecognized, EBV-specific CTL are present in the peripheral blood during acute IM, and include EBNA-1-specific CTL. The importance of accurately defining the in-vivo immune response to EBV is underscored by the ever-growing list of EBV associated malignancies. In addition to providing insights into the oncogenesis and potential treatment of NPC, a newly described link between precursor lesions and EBV infection raises the possibility that heightened immunity to EBV or EBV-infected cells may prevent the development of NPC. An obvious expectation would include extension of such knowledge to other EBV associated malignancies such as B and T cell lymphomas, Hodgkin's lymphomas, and smooth muscle tumors. First however, existing gaps in knowledge regarding the immune response to EBV and EBV-associated malignancies must be closed. Details about the viral gene products which are involved in stimulating a broadly protective, virus-specific immune response in a large number of individuals is fundamental to the design of an effective EBV vaccine. Since the presence of activated CD8+ T cells correlates with the rapid decline of EBV infected B cells in the peripheral blood, a concise description of the EBV-specific CTL response in the setting of acute infection will be necessary for the rational design of an effective acute IM vaccine. Increased understanding of viral escape mechanisms is also likely to contribute to therapeutic modalities to treat autoimmune disorders.

Influenza A virus infections are a major cause of morbidity and mortality in the United States and throughout the world. The current vaccine elicits primarily a humoral response that is specific for the external glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, these are the viral proteins that are most susceptible to antigenic shift and drift, and can evade the humoral response. Cytotoxic T lymphocytes (CTL) recognize and lyse virus-infected cells and are important in clearing influenza A virus infections. CTL can recognize epitopes on both the external glycoproteins and the more conserved internal viral proteins. This thesis investigates the hypothesis that there is a broad CTL memory response in humans, and, if boosted by vaccines, these CTL may help clear influenza A virus strains of different subtypes. The CTL repertoire specific for influenza A viruses reported in inbred mice is extremely limited and has focused on a few immunodominant epitopes. We perfonned preliminary bulk culture chromium release assays using human peripheral blood mononuclear cells (PBMC) stimulated with influenza virus strain A/PR/8/34 (H1N1) in vitro. CTL activity was observed against autologous B-lymphoblastoid cell lines (B-LCL) infected with vaccinia constructs that expressed several influenza A viral proteins, including nucleoprotein (NP), matrix (M1), nonstructural 1 (NS1) and polymerase (PB1). This was more diverse than the limited response reported in inbred mice. To further characterize the CTL repertoire in humans, PBMC from healthy adult donors were stimulated and CTL were cloned by limiting dilution. Isolated cell lines were further characterized by their CD4/CD8 surface expression, histocompatibility leukocyte antigen (HLA) restriction, cross-reactive or subtype-specific influenza A subtype recognition, and epitope recognition. CTL lines isolated from three donors recognized epitopes on many different influenza virus proteins. The ELISPOT assay was used to identify the number of IFN-γ- secreting cells and determine the precursor frequency of the CTL specific for the epitopes that were mapped. The precursor frequency of IFN-γ producing CTL ranged from 1 in 4,156 PBMC to 1 in 31,250 PBMC. The precursor frequency for one epitope was below the level of detection of this assay, but most of the memory CTL were readily detected. The cross-reactive or subtype-specific recognition of various human influenza A subtypes by these T cell lines was determined by chromium release assays. Most of the CTL lines recognized B-LCL infected with any of the three influenza A subtypes that have caused epidemics in the last century (H1N1, H2N2, and H3N2) and recognized epitopes on conserved internal influenza viral proteins. Most of the subtype-specific cell lines recognized the surface HA or NA glycoproteins, which are not well conserved between influenza subtypes. Although most of the T cell lines that were characterized were cross-reactive with influenza viruses of human origin, infection of humans with a divergent swine or avian derived strain could cause a global pandemic. To study the human CTL responses to non-human influenza viruses, B-LCL were infected with an Hsw1N1 influenza A virus of swine origin, and cell lines were tested for recognition of these targets in a chromium release assay. Most cell lines lysed the targets infected With the Hsw1N1 subtype to the same degree as targets infected with the human H1N1 strain. Two influenza viruses of duck origin were also tested and were recognized by many of the cell lines. The subtypes of these duck strains were Hav1N1 and H5N2. The isolates of influenza A virus from the Hong Kong outbreak of 1997 were also used to infect targets and analyze recognition by these CTL. We found that approximately 50% of the human T cell lines tested recognized both of the Hong Kong isolates, 25% recognized at least one isolate, and 25% recognized neither isolate to the same degree as the A/PR/8/34 (H1N1) virus. We analyzed the amino acid (aa) changes in the epitopes of the T cells lines from the 25% of cell lines that did not recognize either Hong Kong virus isolate. Non-conservative mutations were found in all of the epitopes that lost recognition by the human CTL lines. Bulk cultures of PBMC from three donors that were stimulated with A/PR/8/34 (H1N1) influenza A virus of human origin recognized all of the non-human virus strains tested. Thus, humans have memory CTL that recognize influenza viruses of avian and swine species. This may provide a second line of defense against influenza infection in case of exposure to a novel influenza A virus derived from these species. These results made it clear that humans have broad CTL memory to influenza A virus. In order to determine whether these T cells could be boosted in a vaccine, immune-stimulatory complexes (Iscom) incorporating inactivated influenza particles were tested in vitro. Iscoms containing inactivated influenza A vaccine (Flu-Iscom) were used to pulse autologous B-LCL overnight that were then used as targets in chromium release assays with human CTL lines as effectors. A CD8+ HA-specific CTL line lysed these targets, but not targets pulsed with Iscoms alone or with inactivated influenza A vaccine alone. An NS1-specific cell line recognized targets pulsed with NS1 protein and Iscoms, but not targets pulsed with Iscoms or NS1 protein alone. Therefore, CTL could recognize in vitrotarget cells that were exposed to the Iscom vaccines containing their specific epitope. Flu-Iscom and Iscom mixed with inactivated influenza virus particles (Flu-Iscomatrix) were then used as vaccines in a clinical trial to test CTL and neutralizing antibody induction against influenza. Fifty-five donors were bled pre-vaccination, and on days 14 and day 56 post-vaccination. Bulk culture chromium release assays were performed using targets infected with live vaccine strain viruses. There were significantly more increases in the influenza A specific CTL activity in the PBMC of donors that were vaccinated with the Flu-Iscom and Flu-Iscomatrix vaccines than in recipients of the standard vaccine. In order to determine whether these increases in cytotoxicity were due to an increase in the precursor frequency of influenza specific CTL, the PBMC were used in ELISPOT assays to assess the changes pre-and post-vaccination. When there was an increase in the level of cytotoxicity detected in bulk culture CTL, there was often also an increase in the precursor frequency of influenza-specific CTL. Peptide-specific increases in the number of CTL that recognize epitopes such as M1 aa 58-66 were detected in several donors confirming the increase in influenza-specific CTL post-vaccination. Another type of T cell that may be involved in defense against viruses is the γδ T cell. T cells expressing the γδ T cell receptor (TCR) have been found extensively in mucosal tissues in mice and humans. Influenza A viruses enter via the airway tract, infecting the epithelial cells at the mucosal surface. These epithelial cells have been shown in vitro to be targets for influenza-specific cytolytic recognition of αβ T cells. To analyze whether γδ T cells can respond to influenza A-infected APCs, PBMC were stimulated with influenza A virus. Intracellular IFN-γ staining was used to determine whether γ/δ T cells can secrete IFN-γ in response to the influenza A virus infection. We observed an increase in the percentage of γ/δ T cells secreting IFN-γ post-influenza A virus infection of PBMC compared to uninfected or allantoic fluid-stimulated cultures. These T cells also upregulated CD25 and CD69 in response to live influenza A virus. We focused on the responses in the CD8- population of γδ T cells, which are the majority of γδ T lymphocytes. Furthermore, the increases in IFN-γ production and activation marker expression were much more clear in the CD8- γδ+ T cells. The level of CD8- γδ T cell activation with inactivated influenza A virus was much less, and in some cases no higher than uninfected PBMC. The CD8+ αβ and γδ responses could be partially blocked by anti-class I antibodies, but the CD8- γδ responses could not. Vaccinia virus infection did not activate the CD8- γδ T cells to the same degree as influenza virus infection. γδ T cells are thought to have a regulatory role that includes the secretion of cytokines and epithelial growth factors to help restore tissue back to health. Humans have broad multi-specific T lymphocyte responses by αβ T cells to influenza A viruses and those responses are cross-reactive with human, avian, and swine virus strains. These CTL can be activated in vitro and boosted in number in vivo by Iscom incorporating vaccines. There is also a population of γδ+ T lymphocytes in humans that responds to influenza virus infection by producing cytokines and becoming activated. Increasing memory CTL as a second line of defense against influenza A viruses may be important in future vaccine development.

Magister Legum - LLM / Corruption is a serious problem which has many negative impacts on sustainable economic development globally. The clandestine nature of corruption makes it difficult to detect. Hence, efforts to combat corruption successfully demand comprehensive anti-corruption legislation, strong powers, as well as special investigative techniques and strategies. An effective anti-corruption regime requires a comprehensive anti-corruption legal framework which not only punishes all forms of corruption but also capacitates anti-corruption institutions. A strong anti-corruption agency is a crucial requirement and a necessary part of a country's anti-corruption strategy. The failure or the success of an anti-corruption agency depends on a variety of factors, such as powers and means to detect, investigate and prosecute corruption and related offences. The lack of trained staff, as well as the lack of adequate material resources, also affects the effectiveness of an anti-corruption agency. The anti-corruption agencies covered by this research are not empowered or resourced sufficiently, which may result in their ineffectiveness. This paper provides a set of recommendations in respect of the powers and strategies needed for a successful anti-corruption agency. / South Africa