Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2008 Nov 30

Thesis (Ph. D.)--Georgia State University, 2006. / Title from title screen. Dabne White Dixon, committee chair; Kathryn Betty Grant, Markus W. Germann, committee members. Electronic text (252 p. : ill., charts (some col.)) : digital, PDF file. Description based on contents viewed June 18, 2007. Includes bibliographical references.

A newly discovered enteric virus has recently been associated with large outbreaks of waterborne gastroenteritis. Most commonly referred to as the Norwalk agent, this virus appears to be morphologically and biophysically similar ·to the parvoviruses. Presently there is very little known about the fate of parvoviruses in environmental systems. In this study the parvovirus H-1, a putative human virus containing single-stranded DNA (ssDNA), was used as a model virus for chlorine inactivation experiments. The purpose of this research was two-fold: first, to investigate the kinetics of inactivation of parvovirus H-1 by low levels of free chlorine (0.05 - 0.20 mg L⁻¹) at pH 7 and at 5, 10, 20, and 30°C; and secondly, to determine the mechanism by which chlorine inactivates this virus. Inactivation occurred in the usual dose-response relationship, that is, increasing the chlorine dose caused an increase in the rate of inactivation. The results indicated that perhaps more than one reaction mechanism was responsible for inactivation, and the reaction mechanism was a function of temperature. The energy required for the inactivation reaction using 0.05 mg L⁻¹ free chlorine from 5 to 30°C was graphically determined to be 2.4 Kcal mole⁻¹. The change in entropy was calculated to be -52.34 entropy units. From the mechanism study it was concluded that the initial action of chlorine on parvovirus H-1 was on the capsid. Alterations in the two major capsid proteins caused the virion to rupture, and, as evidenced by electron microscopy the ssDNA was exposed. Also, the adsorption ability of the chlorine-treated virions to host cells was significantly inhibited. This was presumably due to the effect on the spatial arrangement of the capsid proteins in their entirety rather than a loss of, or change, in only one polypeptide. The sedimentation rate of the chlorine-treated whole virus decreased from 116S to 43S. The chlorine caused certain sites on the capsid proteins to become highly reactive which facilitated the formation of higher molecular weight aggregates as detected by fluorographs of electrophoretic protein patterns in polyacrylamide gels. Most significant was the discovery that the ssDNA remained undamaged and was still capable of in vitro replication even after 60 minutes of exposure to 5 mg L⁻¹ of sodium hypochlorite at pH 7. / Ph. D.

Measurement is a vital tool for organizations attempting to increase, evaluate, or simply maintain their overall security posture over time. Organizations rely on defense in depth, which is a layering of multiple defenses, in order to strengthen overall security. Measuring organizations' total security requires evaluating individual security controls such as firewalls, antivirus, or intrusion detection systems alone as well as their joint effectiveness when deployed together in defense in depth. Currently, organizations must rely on best practices rooted in ad hoc expert opinion, reports on individual product performance, and marketing hype to make their choices. When attempting to measure the total security provided by a defense in depth architecture, dependencies between security controls compound the already difficult task of measuring a single security control accurately. We take two complementary approaches to address this challenge of measuring the total security provided by defense in depth deployments. In our first approach, we use direct measurement where for some set of attacks, we compute a total detection rate for a set of security controls deployed in defense in depth. In order to compare security controls operating on different types of data, we link together all data generated from each particular attack and track the specific attacks detected by each security control. We implement our approach for both the drive-by download and web application attack vectors across four separate layers each. We created an extensible automated framework for web application data generation using public sources of English text. For our second approach, we measure the total adversary cost that is the total effort, resources, and time required to evade security controls deployed in defense in depth. Dependencies between security controls prevent us from simply summing the adversary cost to evade individual security controls in order to compute a total adversary cost. We create a methodology that accounts for these dependencies especially focusing on multiplicative relationships where the adversary cost of evading two security controls together is more than the sum of the adversary costs to evade each individually. Using the insight gained into the multiplicative dependency, we design a method for creating sets of multiplicative security controls. Additionally, we create a prototype to demonstrate our methodology for empirically measuring total adversary cost using attack tree visualizations and a database design capable of representing dependent relationships between security controls.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Not available / text

Thesis (Ph.D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Sept. 18, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology, University of Alberta." Includes bibliographical references.

Ribavirin, a synthetic nucleoside, was found to inhibit the replication of Rous sarcoma viruses (RSV) and subsequent cell transformation in chick embryo fibroblasts (CEF). It also blocked the transformation of normal rat kidney (NRK) cells infected with temperature-sensitive mutants of RSV. The action of Ribavirin was found to be reversible as removal of the drug from the NRK cells reversed the effects on cell transformation. Ribavirin appears to have a static effect on cell growth of both NRK and CEF cells. In addition, guanosine, xanthosine and inosine altered the effect of Ribavirin on cell growth.

Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain. Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules. Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses. In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs. Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process. Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA- E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells. HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells. In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.

Indiana University-Purdue University Indianapolis (IUPUI) / Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.