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In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)

The den V gene from bacteriophage T4 codes for a small, pyrimidine dimer specific, endonuclease. Recent studies have shown that transfection of the gene into DNA excision repair deficient, Xeroderma Pigmentosum cells, can partially restore the excision repair ability of the cells and results in an increased resistance to UV light. In this study the den V gene has been inserted into Herpes Simplex Virus type 1 (HSV 1) in order to determine if HSV 1 can be used as a suitable vector for studying DNA repair genes. A 1.9 kb cartridge containing the den V gene, the 3' LTR of Rous Sarcoma Virus as the promoter, and the SV40 polyadenylation signals was inserted as the thymidine kinase locus of the virus. Properly initiated transcription form the construct, HDV 1, was verified by primer extension analysis. The Host cell reactivation of this virus and several other strains of HSV 1 were examined in normal and Xeroderma Pigmentosum cells. The results from these experiments suggest that both the viral DNA polymerase and thymidine kinase genes play important roles for the survival of UV irradiated HSV 1. / Thesis / Master of Science (MS)

Many viral transcriptional activators have been shown to activate genes of heterologous systems. To assess the ability of the herpes simplex virus ICP4 trans-activating protein to complement an adenovirus mutant lacking its own trans-activator, the E1a protein, I constructed an adenovirus type 5 vector containing a temperature sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in human cells which are permissive (293) or nonpermissive (KB and R970-5) for E1a⁻ viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus, however it is incapable of complementing adenovirus E1a⁻ mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. My results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators. / Thesis / Master of Science (MS)

Herpes simplex virus (HSV) infections are common in all human populations and for most people they represent relatively mild lifelong infections. To facilitate the persistent infection of hosts, HSV has evolved immune evasion strategies which suppress various aspects of the immune response including the actions of complement and antibodies. Previously in our laboratory, an HSV immediate early protein called ICP47 was shown to inhibit the MHC class I antigen presentation pathway and thereby block recognition of virus infected cells by CD8+ cytotoxic T lymphocytes (CTL). This thesis explores the potential cellular targets of ICP47. Using immunoprecipitation I found ICP47 associates with the transporter associated with antigen presentation (TAP). By blocking the transport of peptide antigens into the endoplasmic reticulum, MHC class I molecules become unstable and are subsequently degraded before displaying HSV antigens on the cell surface. Thus, CTL destruction of cells infected with HSV is blocked. In addition, an interaction between an ICP47 bacterial fusion protein, called GSTICP47-1 and a cellular protein, calcyclin, was examined. The functions of calcyclin are largely unknown. However, based on its association with ICP47, it was possible that calcyclin might play a role in the class I pathway -perhaps as the peptide shuttle. Nevertheless, the results of several experiments were consistent with the notion that calcyclin and ICP47 may not interact in vivo and that calcyclin may not play a role in the MHC class I antigen presentation pathway. / Thesis / Master of Science (MS)

Host cell reactivation (HCR) of UV-irradiated (UV'd) herpes simplex virus type 2 (HSV-2), capacity of UV'd cells to support HSV-2 plaque formation and UV enhanced reactivation (UVER) of UV'd HSV-2 were examined in human fibroblasts. The cells were derived from four Cockayne's Syndrome (CS) patients, 5 xeroderma pigmentosum (XP) patients and 5 normal patients. Survival curves for HCR of HSV-2 plaque formation showed 2- components. HCR was not significantly different in the CS strains and an XP variant strain compared to normal, whereas all excision deficient strains showed a significant reduction in HCR. The o37 values for the delayed capacity curves were in the range 8.6-12.4 J/m2 for the normal strains, 3.1-5.1 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.40-1.98 J/m2 for the XP excision deficient strains examined. UVER was also examined for HSV-2 UV-irradiated to survival levels of 10-2 and 10-3 in unirradiated cells. Maximum delayed UVER was observed in normal strains at a UV dose of 15 J/m2 to the virus. Maximum UVER in CS cells was detected at a UV dose of 5 J/m2 to the cells, in XP excision deficient cells maximum UVER occurred at doses ranging from 0.5-2.5 J/m2 to the cells, and in XP variant maximum UVER occurred at 10 J/m2 to the cells. In all cell strains the level of UVER increased with increasing UV dose to the virus. Results are discussed in terms of the repair defects of CS and XP cells and their relationship to possible viral repair functions. In addition, the possible existence of an inducible DNA repair response is discussed in terms of the results of this study. / Thesis / Master of Science (MSc)

Urban travel demand forecasting and traffic assignment models are important tools in developing transportation plans for a metropolitan area. These tools provide forecasts of urban travel patterns under various transportation supply conditions. The predicted travel patterns then provide useful information in planning the transportation system. Traffic assignment is the assignment of origin-destination flows to transportation routes, based on factors that affect route choice. The urban travel demand models, developed in the mid 1950s, provided accurate and precise answers to the planning and policy issues being addressed at that time, which mainly revolved around expansion of the highway system to meet the rapidly growing travel demand. However, the urban transportation planning and analysis have undergone changes over the years, while the structure of the travel demand models has remained largely unchanged except for the introduction of disaggregate choice models beginning in the mid-1970s. Legislative and analytical requirements that exceed the capabilities of these models and methodologies have driven new technical approaches such as TRANSIMS. The Transportation Analysis and Simulation System, or TRANSIMS, is an integrated system of travel forecasting models designed to give transportation planners accurate, and complete information on traffic impacts, congestion, and pollution. It was developed by the Los Alamos National Laboratory to address new transportation and air quality forecasting procedures required by the Clean Air Act, the Intermodal Surface Transportation Efficiency Act, and other regulations. TRANSIMS includes six different modules: Population Synthesizer, Activity Generator, Route Planner, Microsimulator, Emissions Estimator, and Feedback. This package has been under development since 1994 and needs significant improvements within some of its modules. This dissertation enhances the interaction between the Route Planner and the Microsimulator modules to improve the dynamic traffic assignment process in TRANSIMS, and the Emissions Estimator module. The traditional trip assignment is static in nature. Static assignment models assume that traffic is in a steady-state, link volumes are time invariant, the time to traverse a link depends only on the number of vehicles on that link, and that the vehicle queues are stacked vertically and do not traverse to the upstream links in the network. Thus, a matrix of steady-state origin-destination (O-D) trip rates is assigned simultaneously to shortest paths from each origin to a destination. To address the static traffic assignment problems, dynamic traffic assignment models are proposed. In dynamic traffic assignment models, the demand is allowed to be time varying so that the number of vehicles passing through a link and the corresponding link travel times become time-dependent. In contrast with the static case, the dynamic traffic assignment problem is still relatively unexplored and a precise formulation is not clearly established. Most models in the literature do not present a solution algorithm and among the presented methods, most of them are not suitable for large-scale networks. Among the suggested solution methodologies that claim to be applicable to large-scale networks, very few methods have been actually tested on such large-scale networks. Furthermore, most of these models have stability and convergence problem. A solution methodology for computing dynamic user equilibria in large-scale transportation networks is presented in this dissertation. This method, which stems from the convex simplex method, routes one traveler at a time on the network and updates the link volumes and link travel times after each routing. Therefore, this method is dynamic in two aspects: it is time-dependent, and it routes travelers based on the most updated link travel times. To guarantee finite termination, an additional stopping criterion is adopted. The proposed model is implemented within TRANSIMS, the Transportation Analysis and Simulation System, and is applied to a large-scale network. The current user equilibrium computation in TRANSIMS involves simply an iterative process between the Route Planner and the MicroSimulator modules. In the first run, the Route Planner uses free-flow speeds on each link to estimate the travel time to find the shortest paths, which is not accurate because there exist other vehicles on the link and so, the speed is not simply equal to the free-flow speed. Therefore, some paths might not be the shortest paths due to congestion. The Microsimulator produces the new travel times based on accurate vehicle speeds. These travel times are fed back to the Route Planner, and the new routes are determined as the shortest paths for selected travelers. This procedure does not necessarily lead to a user equilibrium solution. The existing problems in this procedure are addressed in our proposed algorithm as follows. TRANSIMS routes one person at a time but does not update link travel times. Therefore, each traveler is routed regardless of other travelers on the network. The current stopping criterion is based only on visualization and the procedure might oscillate. Also, the current traffic assignment spends a huge amount of time by iterating frequently between the Route Planner and the Microsimulator. For example in the Portland study, 21 iterations between the Route Planner and the Microsimulator were performed that took 33:29 hours using three 500-MHZ CPUs (parallel processing). These difficulties are addressed by distributing travelers on the network in a better manner from the beginning in the Route Planner to avoid the frequent iterations between the Route Planner and the Microsimulator that are required to redistribute them. By updating the link travel times using a link performance function, a near-equilibrium is obtained only in one iteration. Travelers are distributed in the network with regard to other travelers in the first iteration; therefore, there is no need to redistribute them using the time-consuming iterative process. To avoid problems caused by link performance function usage, an iterative procedure between the current Route Planner and the Microsimulator is performed and a user equilibrium is found after a few iterations. Using an appropriate descent-based stopping criterion, the finite termination of the procedure is guaranteed. An illustration using real-data pertaining to the transportation network of Portland, Oregon, is presented along with comparative analyses. TRANSIMS framework contains a vehicle emissions module that estimates tailpipe emissions for light and heavy duty vehicles and evaporative emissions for light duty vehicles. It uses as inputs the emissions arrays obtained the Comprehensive Modal Emissions Model (CMEM). This dissertation describes and validates the framework of TRANSIMS for modeling vehicle emissions. Specifically, it identifies an error in the model calculations and enhances the emission modeling formulation. Furthermore, the dissertation compares the TRANSIMS emission estimates to on-road emission-measurements and other state-of-the-art emission models including the VT-Micro and CMEM models. / Ph. D.

The contrasting infection strategy of herpes simplex virus (HSV) consists of an initial primary lytic infection in epithelial cells, followed by establishment of lifelong latency in sensory and autonomic neurons of the peripheral nervous system that innervate the site of infection. Any cellular stress trigger, ranging from external stimuli such as UV radiation or nerve injury to psychological and physiological stress, can reactivate HSV from latency in the neurons, resulting in recurrent disease episodes. Stress hormones and deprivation of neurotrophic factor (NTF) both have a strong correlation with HSV reactivation from neurons. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. This dissertation provides new understanding of HSV latency and reactivation in response to two orthogonal stress stimuli, viz. stress hormones epinephrine (EPI) and corticosterone (CORT), as well as NTF deprivation that simulates a nerve injury in primary neuronal cultures. In this dissertation, we demonstrate that physiological stress hormones EPI and CORT differentially regulate HSV-1 and HSV-2 reactivation in adult neurons. Both EPI and CORT treatment reactivated only HSV-1 in sympathetic superior cervical ganglia (SCG) neurons, while HSV-2 was reactivated only by CORT in both sensory trigeminal ganglia (TG) neurons and sympathetic superior cervical (SCG) neurons. EPI utilized the combination of α and β adrenergic receptor complex, while CORT signaled through glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) to reactivate HSV in the neurons. NTFs are tissue-target derived growth factors required for neuronal protection and survival. Neurotrophins are also required for maintaining HSV latency, as NTF deprivation reactivates both HSV-1 and HSV-2 in adult sensory TG and sympathetic SCG neurons. In addition, assessing the temporal kinetics of HSV gene expression showed differential expression profiles of viral immediate-early (IE) genes ICP0, ICP4, ICP27 and trans-activator VP16 following treatment with stress hormones and NTF deprivation in HSV-1 and HSV-2 infected neurons. We also show that different molecular mechanisms are involved in HSV latency and reactivation, which are dependent on the stimuli and the type of neurons. Tyrosine kinase receptor-mediated PI3K-Akt-mTORC signaling cascades have been studied for their role in maintaining HSV latency. Activation of β-catenin signalosome expression has also been implicated during HSV latency and following reactivation. GSK3β is a key effector molecule that inter-connects Akt and β-catenin mediated pathways, forming an Akt-GSK3β-β-catenin signaling axis. Analyzing the Akt-GSK3β-β-catenin signaling in response to stress hormone and NTF deprivation revealed significant differences in protein expression levels between HSV-1 and HSV-2 infected sensory and sympathetic neurons. In HSV-1 infected neurons, the Akt-GSK3β-β-catenin maintains the signal transmission in order to keep the neurons alive, but HSV-2 infections obliterated the entire axis in the adult neurons, particularly in sympathetic neurons. In summary, we demonstrate that HSV-1 and HSV-2 do not have a 'one for all' infection mechanism. Establishment of latency and reactivation by HSV is virus specific, stimulus specific and neuron specific. / Doctor of Philosophy / Herpes simplex viruses (HSVs) are common global viral pathogens that are responsible for causing lifelong painful infections and debilitating disease. The two serotypes of HSV include HSV-1, which is associated with oral or ocular disease but can also cause genital disease, and HSV-2, which is predominantly associated with genital herpes. Once infected, both HSV-1 and HSV-2 are present as lifelong reservoirs in our peripheral neurons. Stress stimuli mediated by our stress hormones or external triggers, such as nerve trauma or an axonal injury, can periodically reactivate the latent virus to cause recurrent disease. Clinical manifestation of HSV recurrences range from asymptomatic viral shedding to painful blisters, cold sores, or herpetic keratitis. In some cases, the virus can spread to the central nervous system, causing encephalitis or recurrent meningitis. No vaccines have been approved yet, and the current treatment utilizes nucleoside analogs, such as acyclovir and its prodrug valacyclovir, to ameliorate the symptoms of HSV infection by halting viral replication and if taken as a daily prophylaxis, reduces the chances of clinical recurrence. Given the route and transmission efficiency of HSV, it is practically impossible to prevent herpes infection. To develop strategic therapeutic interventions to lock the virus in its latent phase in the neurons and prevent it from reactivation, a better understanding of neuronal signaling pathways cardinal to HSV latency and reactivation is necessary. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. In this dissertation, we make contributions to better understand HSV latency and reactivation in response to stress stimuli. We show that different stress stimuli exert preferential reactivation between HSV-1 and HSV-2, and are further dependent upon the neurons where they establish latency. Our study specifically focuses on three neuronal stressors that have been associated with HSV recurrences: two stress hormones, epinephrine (EPI) and corticosterone (CORT), as well as deprivation of neurotrophic factors (NTF) that simulates nerve injury. We also focused on a neuronal signaling cascade involved in the response to all of these stimuli, Akt-GSK3β-β-catenin, and viral gene transcripts that respond to these stimuli during reactivation. Comprehensive understanding of the neuronal processes and viral gene transcripts involved during HSV-1 and HSV-2 reactivation in neurons will help the herpes virology field towards development of targeted therapies and vaccines to prevent reactivation and recurrent disease.

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus, often acquired during childhood, that currently infects more than 50% of the human population. The symptoms of infection are herpetic lesions that frequently appear throughout a host's life in response to stress in the orofacial or genital region. As a pathogen, HSV-1 replicates rapidly in epithelial cells, but it is also capable of infecting neurons where it can pursue a lytic or latent infection. Latency is a state of viral quiescence where the virus can persist indefinitely yet remain poised to reactivate. Latency is unique to herpesviruses and key to HSV's success, but the molecular mechanisms that govern this state are unclear. A virus-encoded E3-ubiquitin ligase, Infected Cell protein 0 (ICP0), is often correlated with latency establishment but is detected in opposition to the state of latency. During lytic infection, ICP0 has many biological roles but primarily catalyzes the addition of ubiquitin to target substrate, marking proteins for degradation or altering their function. This ubiquitination ability allows ICP0 to alter the intracellular environment making neurons conducive to lytic or latent HSV-1 infection. ICP0's neuron-specific targets, however, are unknown, representing a significant gap in knowledge. Through the studies presented in this dissertation, we identified some of the neuron-specific ubiquitination targets of ICP0 in neurons. We utilized primary adult sensory neurons of the dorsal root ganglia and HSV-1 viral strains KOS, wild-type virus encoding a fully functional ICP0, and HSV-1 n212, encoding a truncated ICP0 protein, to illuminate the mechanisms involved in establishing and maintaining HSV latency. By using adult primary neurons and functional HSV-1 strains with and without ICP0, we were able to show that ICP0 regulates host and viral proteins during the initial onset of neuronal infection. We also show that based on neuronal conditions set forth before HSV-1 initial infection, host proteins will influence HSV-1 viral proteins to repress viral gene expression, thereby promoting the establishment of latency. / Doctor of Philosophy / Herpes simplex virus (HSV-1) is a virus, often acquired during childhood, that more than 50% of people have. Those who are infected with HSV-1 often have cold sores that appear in response to stress on the face or on the genitals. As a virus, HSV-1 replicates around the eyes, nose, and mouth but can also infect neurons where it can continue to replicate or establish latency. Latency is when the virus is inside the neurons but is unnoticeable and can reappear in response to stress. The state of latency is unique to herpesviruses and key to the success of HSV-1, but scientists are unsure of how it works. A protein made by the virus, Infected Cell Protein 0 (ICP0), is often correlated with the state of latency but is often present when the virus is not latent. ICP0 does a lot to support HSV-1, but it primarily destroys proteins that prevent the virus from replicating. By destroying proteins that prevent HSV-1 replication, ICP0 can help the virus make more viruses. The proteins that are destroyed by ICP0 are currently unknown, which represents a significant gap in knowledge. Through the research conducted in this dissertation, we identified some of the proteins that ICP0 destroys in neurons. We utilized neurons from the dorsal root ganglia and HSV-1 viral strain KOS, which encoded a functional ICP0, and n212, which encodes a nonfunctional ICP0, to study the mechanisms used by the virus to infect neurons. By using HSV-1 viruses with and without ICP0, we were able to show what proteins ICP0 destroys during infection in neurons. We were also able to show that HSV-1's ability to establish latency is dependent on how the neurons handle the initial onset of infection. Overall, a combination of host and viral proteins coordinates the virus's ability to establish latency and persist within a host.