Dietary factors have been implicated in the risk of head and neck squamous cell carcinoma (HNSCC). Much interest has been placed upon the effects of suspected chemopreventative agents found in fruit and vegetables associated with low HNSCC risk. However, studies investigating specific uptake of these agents have failed to show positive results. The possibility exists that single chemopreventative agents fail to provide the same beneficial effect as the various compounds found in a fruits and vegetables is examined. Curatively treated head and neck squamous cell carcinoma patients ingested Juice Plus, a F&V extract supplement containing multiple chemopreventative agents, for 12 weeks. Lymphocyte samples of participants were collect pre- and post- treatment and examined in pairs. Despite the study currently still blinded, surrogate endpoint biomarkers were evaluated to observer any modification between pre- and post treatment samples. Although a paired t-test showed no significant difference between pre- and post treatment samples on surrogate endpoint biomarkers, there is a significant difference in population distribution between treatment times signifying a modification of the surrogate endpoint biomarkers. The exact nature of this difference is pending due to the blinded status of the study.

Objective: The human immunodeficiency virus type 1 (HIV-1) currently infects over 30 million people worldwide. It uses one of two main co-receptors to infect cells. The primary objective of this thesis is to evaluate genotypic and phenotypic assays for co-receptor usage in the clinical setting and investigate approaches for improvement of these assays. Methods: The concordance of recombinant co-receptor phenotyping assays and the predictive ability of genotype-based methods including the ‘11/25’ rule, position specific scoring matrices (PSSMs), and support vector machines (SVMs) were evaluated in the clinical setting using patient-derived plasma samples. Samples and patient data were evaluated in cross-sectional analyses from a retrospective population-based cohort of HIV-infected individuals enrolled in the HIV/AIDS Drug Treatment Program in British Columbia, Canada. Results: Current implementations of HIV V3 region-based predictors for HIV co-receptor usage tested on patient derived samples are inadequate in the clinical setting, primarily due to low sensitivities as a result of difficult to detect minority species. Recombinant phenotype assays also show discordances when tested against each other on the same set of patient derived samples, raising doubts if any of these assays can truly be considered a ‘gold standard’. Significant associations between clinical progression, viral sequence-based predictors of co-receptor usage and the output of recombinant assays are observed, suggesting that sensitivity can be improved by incorporating CD4% into genotype-based predictors. This is verified with a SVM model which showed a 17% increase in sensitivity when CD4% was incorporated into training and testing. Conclusion: This work in this thesis has exposed the difficulty in determining the co-receptor phenotype in the clinical setting, primarily due to minority species. Although genotypic methods of screening for HIV co-receptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turn-around time over phenotypic methods, they are currently inadequate for use in the clinical setting due to low sensitivities. Although the addition of clinical parameters such as CD4 count significantly increases the predictive ability of genotypic methods, the presence of low-levels of X4 virus continues to reduce the sensitivity of both genotypic and phenotypic methods.

A mathematical model of Limiting Dilution Analysis for two limiting parameters is presented and investigated. Limiting Dilution Analysis is a microbiological cell assay developed for immunological application. In the given case we deal with the interaction between B lymphocytes, macrophage derived factor and T-independent antigens. The state of the art is that quantitative statements are only possible if one cell type (in general the B cells) is limiting and all others are in excess present. The basis for this thesis is a set of experiments in which B cells and macrophage derived factor are limiting and all other involved cells and factors are in saturating amounts present. It is shown that so far presented suggestions on modeling Limiting Dilution Analysis for two limiting cell-types are not suitable for this problem. Further, a mathematical model based on data is presented and interpreted in immunological terms with the help of a set of partial differential equations. The basis for the interpretation of the model are changes in affinity and saturation effects, both not incorporated in the so far presented models of the assay. In particular the relevance of mathematical interpretation of this process for the identification of new concepts as the saturation effects is stressed. The model of partial differential equations is highly non-linear but offers the possibility of interpreting the highly interrelated processes apart from each other. / Graduation date: 1994

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

An enzyme-linked immunosorbent assay was developed to measure specific antibody response in bovine sera to bovine adenovirus type 3 (BA3), an etiologic agent of respiratory disease causing economic losses annualy to the cattle industry. Observed endpoint titers were determined using the intersection point from optical density values of serially diluted sera with a positive-negative threshold. Regression equations were determined from standards with titers ranging from low to high and used to predict ELISA titers from a single-serum dilution. A near-linear relationship existed between the observed and predicted ELISA titers of 118 bovine sera (r=0.9261). Predicted ELISA titers were determined using the single-dilution method for another 76 bovine sera and the correlation between the ELISA titers and serum-virus neutralization titers for these sera indicated a strong linear trend (r=0.8172). Both the ELISA and serum-virus neutralization titers on the bovine sera tested indicated widespread exposure to several types of bovine adenovirus. Although detection of active infection would still require examination of sera over time for evidence of a rising titer, the single-dilution ELISA devised should provide a rapid and sensitive method for detection of antibody response to bovine adenovirus type 3. / Graduation date: 1992

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

PXR mediated elevation of CYP3A4 expression is a costly problem in drug development as well as a clinical problem due to clinically important drug interactions caused by the enzyme induction. CYP3A4 is responsible for the metabolism of more than 50% of the drugs commonly used today. Many of these, as well as other compounds e.g. in herbal medicines can induce transcription of CYP3A4 and thereby enhance the metabolism of other drugs, rendering them ineffective or more toxic. By using an in vitro assay for CYP3A4 induction, tests can be performed on candidate drugs early in development and thereby save time and resources since CYP3A4 inducers are eliminated from further development. A reporter gene assay was constructed by inserting three modules, which includes PXR binding sites isolated from the CYP3A4 sequence, in front of a luciferase gene. This construct was transfected together with PXR into HEK 293 cells. Induction was evoked by adding rifampicin, a known CYP3A4 inducer, to the medium. After lysis of the HEK cells and addition of luciferase substrate, luminescence intensity was recorded as a measure of induction. The construct worked and consistently showed induction by rifampicin, but could be further improved to yield higher sensitivity.

The development of non-destructive assay (NDA) capabilities to directly measure the fissile content in spent fuel is needed to improve the timely detection of the diversion of significant quantities of fissile material. Currently, the International Atomic Energy Agency (IAEA) does not have effective NDA methods to verify spent fuel and recover continuity of knowledge in the event of a containment and surveillance systems failure. This issue has become increasingly critical with the worldwide expansion of nuclear power, adoption of enhanced safeguards criteria for spent fuel verification, and recent efforts by the IAEA to incorporate an integrated safeguards regime. In order to address these issues, the use of Self-Interrogation Neutron Resonance Densitometry (SINRD) has been developed to improve existing nuclear safeguards and material accountability measurements. The following characteristics of SINRD were analyzed: (1) ability to measure the fissile content in Light Water Reactors (LWR) fuel assemblies and (2) sensitivity and penetrability of SINRD to the removal of fuel pins from an assembly. The Monte Carlo Neutral Particle eXtended (MCNPX) transport code was used to simulate SINRD for different geometries. Experimental measurements were also performed with SINRD and were compared to MCNPX simulations of the experiment to verify the accuracy of the MCNPX model of SINRD. Based on the results from these simulations and measurements, we have concluded that SINRD provides a number of improvements over current IAEA verification methods. These improvements include: 1) SINRD provides absolute measurements of burnup independent of the operator’s declaration. 2) SINRD is sensitive to pin removal over the entire burnup range and can verify the diversion of 6% of fuel pins within 3σ from LWR spent LEU and MOX fuel. 3) SINRD is insensitive to the boron concentration and initial fuel enrichment and can therefore be used at multiple spent fuel storage facilities. 4) The calibration of SINRD at one reactor facility carries over to reactor sites in different countries because it uses the ratio of fission chambers (FCs) that are not facility dependent. 5) SINRD can distinguish fresh and 1-cycle spent MOX fuel from 3- and 4-cycles spent LEU fuel without using reactor burnup codes.

Hepatocyte growth factor (HGF) is a multifunctional protein, which secrets via Golgi complex after synthesized, and is hydrolyzed into an active heterodimer containing an £\ and a £] chain by extracellular protease. It is known that HGF functions through surface domain of Met, and thus induces mitosis and metastasis. The interaction domain of HGF is believed to be located in the £\-chain. In order to study these findings structurally and functionally, we designed and constructed four different recombinant coding regions of the gene (NK1, NK2, NK3, and NK4) which was then successfully expressed in E. coli. Purification of these four different recombinant proteins with glutathione-agarose column showed that all of the four constructs had been successfully expressed with some degradations. Cell proliferation assay showed that the recombinant proteins inhibited the growth of breast cancer cells to some extent. The assay also showed that GST-NK1 and GST-NK2 were better inhibitors than GST-NK3 and GST-NK4 to the cancer cells. It is concluded that E. coli expression is an appropriate system for achieving functional HGF.

IgA1 (immunoglobulin A1), a predominant immunoglobulin, is at the first defense line against microbial pathogens infection and invasion, to neutralize pathogenic antigens. Some bacterial pathogens, such as Neisseria meningitidis and Haemophilus influenzae, however, secrete site-specific IgA1 proteases to counteract with the human defense system. The protease is capable of cleaving at the hinge region of immunoglobulin A1 to destroy the structure and function of human IgA1, impairing the role of the immunoglobulin from the host defense. The protease has therefore been implicated as a putative virulence factor that contributes to bacterial colonization, but bacterial isolates from patients with invasive diseases contain both positive and negative IgA1 proteases. To clarify the role of IgA1 protease in bacterial infection, this project is designed to reveal the molecular mechanism of the protease in bacterial infection and colonization. To do this, iga genes encoding non-typable H. influenzae type 1, type 3 and Neisseria meningitidis type 3 IgA1 proteases were isolated, sequenced and then expressed in IgA1 protease-negative E. coli BL21 (DE3). The recombinant proteases have been purified to homogeneity using ion exchange chromatography. Comparison of the deduced amino acid sequences from non-typable H. influenzae IgA1 proteases with other published H. influenzae IgA1 protease revealed a high degree of homology. Sequence analysis indicates that both type 1 and type 3 non-typable H. influenzae IgA1 proteases lack £\-protein in comparison with the iga from N. meningitidis. The role of IgA1 protease in relation to deposition and invasion has also been evaluated in human lung carcinoma cell (A549) model. The results suggest that the IgA1 protease plays a role in the adherence of H. influenzae on epithelial cell surface though the best effectiveness varies upon different pathogenic bacterial strains at different concentrations.