Positive-stranded (+)RNA viruses replicate inside cells and depend on many cellular factors to complete their infection cycle. In the meanwhile, (+)RNA viruses face the host innate immunity, such as cell-intrinsic restriction factors that could block virus replication. Firstly, I have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. This study demonstrates that RH30 blocks the assembly of viral replicase complex, the activation of RNA-dependent RNA polymerase function of p92pol and viral RNA template recruitment. In addition, the features rendering the abundant plant DEAD-box helicases either antiviral or pro-viral functions in tombusvirus replication are intriguing. I found the reversion of the antiviral function of DDX17-like RH30 DEAD-box helicase and the coopted pro-viral DDX3-like RH20 helicase due to deletion of unique N-terminal domains. The discovery of the sequence plasticity of DEAD-box helicases that can alter recognition of different cis-acting elements in the viral genome illustrates the evolutionary potential of RNA helicases in the arms race between viruses and their hosts. Moreover, I discovered that Xpo1 possesses an anti-viral function and exports previously characterized cell-intrinsic restriction factors (CIRFs) from the nucleus to the replication compartment of tombusviruses. Altogether, in my PhD studies, I found plant RH30 DEAD-box helicase is a potent host restriction factor inhibiting multiple steps of the tombusvirus replication. In addition, I provided the evidence supporting that the Nterminal domain determines the functions of antiviral DDX17-like RH30 DEAD-box helicase and pro-viral DDX3-like RH20 DEAD-box helicase in tombusvirus replication. Moreover, I discovered the emerging significance of the Xpo1-dependent nuclear export pathway in tombusvirus replication.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and is the most aggressive malignant primary brain tumor in adults. Without treatment, patients are expected to survive an average of three months. Conversely, current treatment regimens only extend survival to 12-14 months. Characteristically, GBM tumors are highly proliferative, invasive and stop responding to treatments relatively fast due to therapy resistance. Interestingly, GBM also exhibits high metabolic activity but manages to maintain a low level of reactive oxygen species (ROS). These ROS neutralization capabilities are sustained by system Xc–, a sodium-independent, electro neutral transporter that is found in the plasma membrane of GBM cells. System Xc– is composed of a regulatory heavy subunit (4F2hc) linked to a 12 transmembrane domain catalytic light chain subunit (xCT) that mediates the uptake of L-cystine into the cell, and L-glutamate out of the cell, at a 1:1 ratio. Imported cystine is quickly reduced to L- cysteine, the rate limiting substrate in glutathione (GSH) synthesis. Glutathione is a major antioxidant in the central nervous system that is responsible for maintaining intracellular redox homeostasis by neutralizing ROS by direct and indirect methods. The function of chemo and radiation therapy is to generate significant levels of ROS that tigger the cell to undergo apoptosis. High intracellular GSH levels in cancer cells are associated with drug resistance and detoxification of alkylating agents such as temozolomide (TMZ). Therefore, system Xc– represents a potential target to reduce glioma cell survival and reduce tumor progression. Sulfasalazine is an FDA approved drug in the treatment of arthritis and Crohne’s disease and has been shown to inhibit system Xc–. In vitro SASP studies demonstrated a strong antitumor potential in preclinical mouse models of malignant glioma. However, two clinical trials using sulfasalazine with standard chemo and radiation therapy to treat GBM patients were terminated due to off-target effects. Both results showed high toxicity and no change in the overall survival of patients. These studies demonstrate the need for a more effective inhibitor of system Xc–. To further elucidate the role of system Xc– in GBM survival, stable xCT knock-down and over-expressing U251 glioma cells were generated. These lines were characterized for survival, proliferation, apoptosis and resistance to oxidative and genotoxic insult. As expected xCT-knockdown cells exhibited lower GSH levels, increased intracellular ROS and markers for apoptosis after oxidative and genotoxic insult. The xCT-over-expressing cells displayed higher levels of GSH, increased resistance to hydrogen peroxide and various chemotherapy drugs including TMZ. An interesting unforeseen result of xCT over-expression in glioma cells was an increase in the metabolic activity as a result of increased mitochondria. Using xCT-modified glioma lines stably, we demonstrate for the first time that system XC– over-expression not only promotes survival under oxidative stress but may also decreases sensitivity to chemotherapy treatment and increase metabolic properties. Therefore, therapeutic manipulation of this transporter either alone or in combination with other treatments may improve clinical outcome in patients diagnosed with GBM.

For over three decades, amphibian populations have been declining across the globe. Emerging infectious diseases are responsible for some of these declines. Ranaviruses have caused die-offs in wild amphibian populations on 4 continents, in 5 Canadian provinces, and in over 25 U.S. states. In order to understand host-pathogen dynamics, it is critical to establish baseline information on species susceptibility and the effects of natural stressors. The goal of my thesis research was to quantify the effects of anuran development and exposure to invertebrate predators on species-specific susceptibility to ranavirus. My experiments were designed in factorial arrangements, and consisted of exposure to ranavirus during different developmental stages or with and without predator cues in a controlled environment. I found that exposure to invertebrate predator cues did not increase susceptibility to ranavirus for 4 anuran species tested. Susceptibility differed among embryo, hatchling, larval and metamorph stages, but trends differed among species and did not follow predictions based on Xenopus laevis immune function. Low susceptibility during the embryo stage was the only consistent development result among species, perhaps owing to protective qualities of the vitelline membrane or mucoidal capsules surrounding the embryo. Across 7 anuran species tested, mean mortality rates ranged from 5 – 100%, with Lithobates sylvaticus and Scaphiopus holbrookii most susceptible. I found that infection rates and viral load were correlated with mortality rates, thus these variables are good indicators of susceptibility to ranavirus. My results indicate that ranaviruses can cause catastrophic natural mortality in some anuran species, and likely play a significant role in local population dynamics. For highly susceptible species, ranaviruses could cause local extirpations that lead to species declines. More information is needed on the role of natural (e.g., co-infection, competition) and anthropogenic stressors in driving ranavirus epizootic events. I encourage natural resource agencies to initiate ranavirus surveillance programs, especially for rare species and fragmented populations. Future studies should take an immunogenetic approach to identifying mechanisms driving susceptibility. Identifying mechanisms associated with ranavirus emergence is fundamental to developing science-based conservation strategies.

To date, few high school based interventions have been shown to have lasting effects on adolescents' health behaviors. The need for health interventions targeting adolescents is underscored by data showing that several health behaviors with significant short and long term adverse effects begin in early adolescence and become progressively more prevalent toward late adolescence. This project tested the efficacy of a novel anatomy based health education curriculum at increasing health knowledge. The course was taught by first year Yale medical students. The curriculum placed emphasis on nutrition, physical activity and infectious disease. Forty Juniors from Career High School visited Yale's anatomy lab once every two weeks for ten hour-long sessions. In addition to visits to the anatomy lab, students completed two class projects, one covered nutrition and the other focused on exercise. Four additional sessions at Career High School were dedicated to the class projects. Pre and post test analysis showed an improvement in health knowledge with a thirteen percentage point improvement on a standardized health knowledge survey. The students' performance was compared to a control cohort of thirty-one students who were not exposed to the curriculum. Students exposed to the curriculum had a nineteen percentage point advantage compared to control students who had not been exposed. Curriculum efficacy as demonstrated by this small cohort validate further testing with larger cohorts and more vigorous controls as well as separate testing to measure changes in health behavior attributable to curriculum exposure.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Felaktigt serienummer 1251. Härtill 4 uppsatser.

Source: Masters Abstracts International, Volume: 47-06, page: 3551. Adviser: Christine Markham. Includes bibliographical references.

Pandemic outbreaks are unpredictable as to their virus strain, transmissibility, and impact on our quality of life. Hence, the decision support models for mitigation of pandemic outbreaks must be user-friendly and operational, and also incorporate valid estimates of disease transmissibility and severity. This dissertation research is aimed at 1) reviewing the existing pandemic simulation models to identify their implementation gaps with regard to usability and operability, and suggesting research remedies, 2) increasing operability of simulation models by calibrating them via an epidemiological model that estimates infection probabilities using viral shedding profiles of concurrent pandemic and seasonal influenza, and 3) developing a testing strategy for the state laboratories, with their limited capacities, to improve their ability to estimate evolving transmissibility parameters. Our review of literature (Aim 1) indicates the need to continue model enhancements in critical areas including updating of epidemiological data during a pandemic, smooth handling of large demographical databases, incorporation of a broader spectrum of social-behavioral aspects, and improvement of computational efficiency and accessibility. As regards the ease of calibration (Aim 2), we demonstrate that the simulation models, when driven by the infection probabilities obtained from our epidemiological model, accurately reproduce the disease transmissibility parameters. Assuming the availability of sufficient disease reporting infrastructure and strong compliance by both infected population and healthcare providers, our testing strategy (Aim 3) adequately supports characterization of real-time epidemiological parameters. Future research on this topic will be aimed at integrating the laboratory testing strategy with our modeling and simulation approach to develop dynamic mitigation strategies for pandemic outbreaks.

CD28 co-stimulation is required for the generation of naturally occurring regulatory T cells (nTregs) in the thymus through Lck-signaling. However, high level of CD28 suppresses the generation of induced Tregs (iTregs) from naïve CD4 T cells, although underlying mechanism(s) has not been defined. Here we investigated the role of CD28-mediated signaling pathways in the suppression of Treg generation. We used a series of transgenic (Tg) mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk. Regardless of exogenous IL-2, strong CD28 costimulation suppressed iTreg generation through Lck signaling. Using a GVHD model to test the role of CD28-mediated iTreg suppression in T cell pathogenicity in vivo, we found that CD28-Lck T cells induced significantly less GVHD than T cells from CD28-WT mice. Furthermore, we found that the recipients of T cells from CD28-Lck mice generated significantly more iTregs than those with T cells from CD28-WT, which contribute to reduced graft-versus-host disease (GVHD) development in recipients of CD28-Lck T cells. These results indicate that CD28 costimulation can negatively regulate Treg generation and may provide an avenue for control of T-cell immunity or tolerance by regulating Tregs using the CD28 signal as a target. We went a step forward and investigated the therapeutic potential of antigen-specific iTregs in the prevention of GVHD. Donor hematopoietic stem cells and mature T cells are transplanted into a lymphopenic host to potentially cure many cancers and hematopoietic diseases like leukemia in bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HCT), but the frequent development of GVHD is the main drawback of this treatment. nTregs suppress the development of GVHD and may spare graft-versus-tumor effect. However, nTregs are a minor (~5%) subpopulation of CD4 helper T cells in healthy individuals, and using in vitro expanded nTregs is a common strategy to test their therapeutic potential in BMT. The concern of in vitro expanded nTregs may include their stability of Foxp3 (master regulatory gene for the development and function of regulatory T cell) expression and suppressive function, survival in vivo, and the non-selective suppression of the pre-activated nTregs. Antigen-specific activation of the regulatory T cells is important for optimal function. In this study, we used an alternative strategy to generate antigen-specific, iTregs and assessed their suppressive potential by comparing their effectiveness in preventing GVHD with polyclonal iTregs. We found that antigen-specific iTregs prevented GVHD lethality in recipients that expressed the target antigen, but were not protective of recipients who did not express the target antigen. Furthermore, antigen-specific iTregs were significantly more efficient than those polyclonal Tregs in the prevention of GVHD. These results reveal the therapeutic potential of antigen-specific iTregs to prevent GVHD efficiently and selectively, and provide the rationale to use antigen-specific iTregs in clinical HCT.

Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) have been extensively studied in the past 30-40 years. Their potent suppressive capacity shown in several pathological and clinical settings, such as cancer and transplantation, has made it evident that better understanding their development and function is critical. Specifically, Tregs play a pivotal role in preventing autoimmunity, graft-versus-host disease (GvHD), and organ graft rejection. We previously demonstrated that germline or induced SH2 domain-containing inositol 5-phosphatase (SHIP) deficiency in the host abrogates GvHD. Here we show that SHIP-deficiency promotes an increase of FoxP3+ cells in both the CD4+CD25+ and the CD4+CD25- T cell compartments with increased expression of Treg-associated markers. Importantly, SHIP-deficiency does not compromise Treg function. Interestingly, like conventional Tregs, SHIP-/- CD4+CD25- T cells are unresponsive to allogeneic stimulators and suppress allogeneic responses by T cells in vitro, and can mediate reduced lethal GvHD in vivo. Thus, SHIP limits the immunoregulatory capacity of CD4+ T cell, particularly in allogeneic settings. SHIP-deficiency expands the number of immunoregulatory cells in both the T lymphoid and myeloid lineages. Here, we examined if these increases are interrelated. Specifically, we found that myeloid specific SHIP-deficiency leads to expansion of both MDSC and Treg numbers. Conversely, T lineage specific ablation of SHIP leads to expansion of Treg numbers, but not expansion of MDSC, indicating an intrinsic role for SHIP in limiting Treg numbers. Interestingly, MDSC lack SHIP expression suggesting that another SHIP-deficient myeloid cell promotes MDSC and Treg expansion. Also, increased levels of G-CSF, a myelopoietic growth factor, in SHIP-/- mice may extrinsically promote MDSC expansion since we found that G-CSF is required for the expansion of splenic MDSC in mice with induced SHIP-deficiency. MDSC consist of two distinct subsets, granulocytic-MDSC (G-MDSC), and monocytic-MDSC (M-MDSC) that differ in morphology, phenotype, suppressive capacity and differentiation potential. Importantly, M-MDSC can further differentiate into dendritic cells, macrophages and preferentially into G-MDSC, in the presence of tumor-derived factors (TDF). The retinoblastoma gene (Rb1), a tumor suppressor gene and central regulator of the cell cycle and differentiation, has been shown to influence monocytic and neutrophilic lineage commitment and to limit myeloproliferative disease. Here, we examined the role of Rb1 in the biology of MDSC subsets in tumor-bearing mice. Firstly, M-MDSC expressed high levels of Rb1 which remained relatively stable in culture with GM-CSF. Conversely, freshly isolated G-MDSC initially expressed undetectable levels of Rb1 that increased over time in culture, which correlated with increased histone acetylation at the Rb1 promoter. This increased Rb1 expression and histone acetylation was accelerated by histone deacetylase inhibitors (HDACi) treatment, suggesting Rb1 expression may be controlled by histone modification. Furthermore, when treated with HDACi, M-MDSC did not differentiate into G-MDSC in culture, even with TDF present. Finally, induced Rb1 deficiency in vivo promoted an expansion of splenic CD11b+Ly6G+Ly6Clo cells, similar to G-MDSC in tumor-bearing mice. Although further studies are required, these results strongly suggest that Rb1, like SHIP, plays a role in MDSC accumulation, particularly G-MDSC in cancer.

Veterinarians often perform controlled experiments in which they inoculate animals with infectious diseases. They then monitor the transmission process in infected animals. The aim of such experiments can be to assess vaccine effects. The fitting of individual-level models (ILMs) to the infectious disease data, typically achieved by means of Markov Chain Monte Carlo (MCMC) methods, can be computationally burdensome. Here, we want to see if a vaccination effect can be identified using simpler regression-type models rather than the complex infectious disease models. We examine the use of various logistic growth curve models, via a series of simulated experiments in which the underlying true model is a mechanistic model of infectious disease spread. We want to investigate whether a vaccination effect can be identified when only partial epidemic curves are observed, and to assess the performance of these models when experiments are run with various sets of observational times.