▼ The purpose of this study is to determine the effectiveness of the See Clearly Method. The See Clearly Method is a program of eye exercises designed to naturally improve eyesight without traditional optical correction. The goals of this study are to quantify changes that occur after using the See Clearly Method for one month, specifically changes in visual acuity, refractive error and subjective quality of life. Thirty subjects were randomized into two groups. The test group was given the See Clearly Method and was instructed to use the kit for one month, keeping track of progress in a daily journal. The control subjects did not use the See Clearly Method during the month. Uncorrected high contrast visual acuity, subjective refraction and autorefraction were measured at baseline and at one month and analyzed for change. Subjective quality of life was assessed using the National Eye Institute Refractive Error Quality of Life (NEI-RQL) instrument both at baseline and at one month. No statistically significant changes were found for any of the outcomes analyzed, which indicates that in this study, the See Clearly Method did not change the patient's refractive error or uncorrected visual acuity, and it did not improve visual quality of life. Findings can be used to educate doctors and patients about this product Advisors/Committee Members: Zadnik, Karla.

▼ Years of clinical and experimental evidence have shown that both the antigen-nonspecific innate immune system and the antigen-specific adaptive immune system can effectively eliminate malignant cells that remain after front-line therapy for cancer. Because the immune response to any given stimulus requires the coordinated activity of a large number of diverse cell types, elaborate communication networks have evolved that utilize direct cell-cell interactions as well as soluble growth factors, or cytokines, that can potentially travel great distances in the body. Knowledge of the mechanisms and effects of these cell-cell and cell-cytokine-cell interactions is of paramount importance as physicians and scientists advance the frontiers of cancer immunotherapy. Presented here is a series of studies that define roles for the cytokine interleukin 15 (IL-15) in acute graft versus host disease (GVHD) and graft rejection. Mediated by both the innate and adaptive immune systems, graft rejection and acute GVHD are the most common life threatening side effects of allogeneic bone marrow transplantation (BMT), a promising immunotherapeutic approach for aggressive and otherwise incurable hematopoietic malignancies. Also presented are studies evaluating a novel therapeutic antibody designed to interrupt the cell-cell signals that serve to prevent tumor cell lysis by natural killer (NK) cells, a critical part of the innate immune system. Together, these data unravel a small portion of the complex interactions between immune effector cells and malignant cells and provide justification for future basic and clinical immunotherapeutic studies. Advisors/Committee Members: Caligiuri, Michael A.

▼ In this dissertation, statistical mechanics, graph theory, and machine learning methods have been used to study the topology, modularity, organization and robustness of the proteome network of Saccharomyces cerevisiae. The protein-protein interaction dataset is obtained by combining high confidence interactions, and is validated from multiple perspectives. Statistical mechanics is then used to analyze the connectivity distribution, graph spectrum, shortest path distance and clustering coefficients of the network, which indicates that the network is both scale-free and modular. Microarray gene expression profiles are used to compute the weight for each interaction and the network is represented as a weighted graph. An edge betweenness-based algorithm is developed and applied on the graph, and a set of functional modules is identified. The functional modules are then validated rigorously against gene annotation, growth phenotype and protein complexes. It is found that genes in the same functional module exhibit similar deletion phenotype, and that known protein complexes are largely contained in the functional modules. Studies on the relationship between the gene expression profiles of hubs and their interacting proteins indicate that subpopulations of hubs exist in the yeast proteome network, which are classified as core, local and global hubs. By examining these hub populations from the perspectives of protein complexes, interaction overlap, clustering coefficients, module connectivity, and visualization, it is found that global hubs form the backbone of module-module interaction, while core hubs are organizers within functional modules. In addition, it is found that each hub type preferentially interacts with hubs from the same population, which suggests an ordered architecture for the network. Studies on gene expression changes suggest that global hubs are the major and early responders in cellular response. Next, network breakdown simulation and graph spectrum are used to examine the contributions of each hub population to the robustness of the yeast proteome network. The results indicate that network organizers contribute most to the robustness at both global and local levels. And last, it is found that genes contributing most to the robustness of functional modules, not that of the entire network, are more likely to be essential. Advisors/Committee Members: Yuan, Bo.

▼ Ultraviolet light B (UVB) is a ubiquitous complete carcinogen of the skin. Due to their immunosuppressive therapy, organ transplant recipients have a 60-250 fold increased likelihood to develop aggressive, highly metastatic UVB-induced squamous cell carcinomas (SCC) compared to the general population. This thesis examines both UVB-induced cutaneous inflammation and UVB-induced skin tumor models mimicking transplant immunosuppression by depleting systemic CD4+ or CD8+ T lymphocytes. Our hypothesis is that a systemic decrease of CD4+, but not CD8+ T lymphocytes will increase the amount of UVB-induced DNA damage in acute UVB exposure and result in increases in tumor number following chronic UVB exposure.In the UVB-induced inflammation model, a decrease in systemic CD4+ but not CD8+ T lymphocytes increased UVB-induced neutrophil number and activity in the skin. Both T lymphocyte depletion groups showed increased DNA damage through increased levels of p53+ epidermal cells. This increase in DNA damage was not due to an increase in direct UVB-induced damage, as seen by immunohistochemical analysis of cyclobutane pyrimidine dimers (CPDs). Increased UVB-induced DNA damage could increase the amount of UVB-induced skin cancer that develops. The anti-inflammatory, anti-angiogenic drug, celecoxib, was able to almost completely abate the exacerbated UVB-induced inflammatory reaction in the CD4-depleted animals.In the chonic UVB-induced skin tumor model, a decrease in systemic CD4+ but not CD8+ T lymphocytes significantly increased tumor numbers. Tumors isolated from CD4-depleted mice also showed increased invasive properties and increased numbers of p53 point mutations. Topical treatment with the anti-inflammatory drug celecoxib significantly decreased tumor numbers in control, CD4- and CD8-depleted mice. Based upon our findings, it appears that there are multiple mechanisms by which systemic immunosuppression can cause an increase in tumor number and aggressiveness in transplant recipients. Our studies suggest that clinically blocking increased levels of inflammation in the skin via the topical application of celecoxib as a chemo-preventative agent might be one potential mechanism to decrease the increased risk of skin cancer observed in immunosuppressed patients. Advisors/Committee Members: Oberyszyn, Tatiana M.

▼ Type I Diabetes Mellitus results from autoimmune destruction of insulin-producing pancreatic islet beta cells, leading to loss of normal glucose homeostasis. Transplantation of functional pancreatic islets is a potential cure for type I diabetes. Despite recent clinical advances, application is limited due to progressive functional deterioration late after transplantation. Causes of late islet allograft dysfunction are unknown but may represent undiagnosed immunologic damage to islets. Heterotopic islet transplantation to the liver has yielded the best clinical outcomes. However, the liver is a metabolically and immunologically active organ, and experimental islet transplant studies have not historically accounted for potential effects of this immune locale on islet allograft survival and function. This dissertation research was designed to test the hypothesis that late loss of islet allograft function is due to immunologic barriers, which arise due to the activation of immune responses particular to the microenvironment of the liver. The goals of these studies were: (1) to design a clinically relevant experimental model of pancreatic islet transplantation, (2) to determine the influence of immune locale on acute alloimmune responses elicited by allogeneic islets, (3) to analyze the susceptibility of islet allografts to late immune damage following successful engraftment, and (4) to determine the utility of peripheral immune monitoring to predict transplant outcome. A clinically relevant model of intrahepatic islet transplantation was developed and was validated both functionally and histologically. Transplantation of islets to the liver neither altered the phenotype of acute rejection nor prevented induction of long-term islet acceptance. Development of a model for late allospecific immune activation (by sequential donor-matched allogeneic hepatocyte transplantation) demonstrated that islets remain vulnerable to damage by activation of local, but not peripheral, alloimmune responses. Late immune damage results from local activation of (CD4-independent) CD8+ T cell responses. However, novel immunotherapy targeting both CD4-dependent and CD8-dependent immune responses protects islets from late immune damage. Finally, peripheral monitoring to detect activation or suppression of donor-reactive humoral (alloantibody) and cell-mediated (DTH and in vivo allospecific cytotoxicity) immune responses predicted the immune status of islet allografts in vivo. Advisors/Committee Members: Bumgardner, Ginny Li.

▼ The results of numerous basic studies support the use of anti-CTLA-4 antibodies in human cancer therapy. To assist in the translation of this concept to the clinic it would be helpful to establish preclinical models to identify anti-human CTLA-4 antibodies that can induce anti-cancer immunity with acceptable autoimmune side effects. To that end we have produced a human CTLA-4 knock-in mouse in which murine CTLA-4 has been replaced with its human homolog. We used our knock-in mouse to screen a panel of anti-human CTLA-4 antibodies to determine whether our model was useful in discriminating the antibodies’ therapeutic effects and autoimmune side-effects. Surprisingly, while all of the antibodies induced protection against cancer and demonstrated some autoimmune side effects, the antibody that induced the strongest protection also induced the least autoimmune side effects. These results suggest that anti-tumor immunity is not necessarily linked to autoimmunity, and that it may be possible to uncouple the two. Studies of neonatal tolerance have suggested that pre-existing antigens are tolerogenic to subsequently generated T cells. However whether the same is true in the adult host is not known. As the effect of tumor on developing T cells has not been tested, we analyzed the impact of resident tumor on the development of tumor-reactive T cells in the thymus and their immune competence in the periphery. Our results suggest that newly produced T cells with specificity for pre-existing tumor cells are activated rather than inactivated by tumor antigens in the host. The importance of B7:CD28 costimulation in Treg development is a widely accepted, yet poorly understood concept. As Treg have been shown to be produced in the periphery as well as intrathymically, it remains unclear as to whether costimulation plays a similar role and produces equivalent effects at each location. To further explore this matter we characterized the expansion of regulatory T cells within Treg-deficient mice following agonistic anti-CD28 antibody treatment. We show here, in the thymus costimulation promotes Treg expansion by licensing developing thymocytes, as Treg are generated de novo, in the absence of proliferation. In the periphery costimulation promotes Treg expansion primarily through enhanced proliferation. Advisors/Committee Members: Liu, Yang.

Advisors/Committee Members: Hitchcock, F.A.

Advisors/Committee Members: Lanese, Richard R.

Advisors/Committee Members: Ellingson, Harold V.

▼ We have utilized a newly developed F11 generation of Advanced Intercross Line (AIL) mouse population to fine map three mouse Pulmonary adenoma susceptibility (Pas) loci: Pas1 (on chromosome 6), Pas2 (on chromosome 17) and Pas3 (on chromosome 19). By selectively genotyping 30% of the population, we have confirmed the Pas1 QTL and refined it into an interval of approximately 1.0 cM (~1.3 Mb) in the vicinity of the Kras2 gene. The Pas2 QTL was detected by both ANOVA and regression analysis but not by Mapmaker software. An interaction between the Pas1 and Pas2 QTLs was also revealed. However, the Pas3 QTL has not been confirmed in this study. On the other hand, congenic strategy was also utilized to fine map the Pas1 locus. A set of new polymorphic markers has been developed to assist this fine-structure mapping. Combining results from the AIL project and the congenic project, we have refined the Pas1 QTL into a less than 1-Mb minimum candidate region based on the Celera mouse genome map encompassed by the markers D6Osu6 and D6Osu11. There are 24 putative genes located within this region. Candidate gene screening for this region has presented the Lrmp/Jaw1, Ak016641/Pas1c1 (for Pas1 candidate 1) and a novel gene, Pas1c2 (for Pas1 candidate 2) as strong candidates for the Pas1 QTL, and they will be further tested by in vitro and in vivo functional analyses. Using a similar congenic strategy, the Pulmonary adenoma resistance 2 (Par2) QTL has been narrowed to an approximately 6.3-Mb region flanked by the marker D18Mit103 and the marker D18Mit162. Four genes are possible Par2 candidates based on real-time PCR analyses, including Myo5b, Smad7, Mapk4, and GABA-A receptor-like gene mCG58197. Sequencing analyses for this region found that the Rad30b gene, encoding the DNA–dependent polymerase iota (Polé), carries 25 nucleotide polymorphisms in its coding region between the A/J and BALB/c strains, leading to ten amino-acid alterations. Functional analyses on the above five genes will be needed to clarify their Par2 candidacy. In addition, computational Single Nucleotide Polymorphism (SNP) analyses revealed that 54 putative genes carried various types of SNPs when comparing genomic sequences from A/J, 129X1/svJ, 129S1/svImJ, DBA/2J, and C57BL/6J mice. Screening of these SNPs in the future may help us find new polymorphic markers and identify new Par2 candidate genes. Advisors/Committee Members: You, Ming.