The role of intracellular calcium perturbations in muscle damage and dysfunction in mouse models of muscular dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. DMD is clinically characterized by severe, progressive and irreversible loss of muscle function, in which most patients lose the ability to walk by their early teens and die by their early 20’s. Impaired intracellular calcium (Ca2+) regulation and activation of cell degradation pathways have been proposed as key contributors to DMD disease progression. This dissertation research consists of three studies investigating the role of intracellular Ca2+ in skeletal muscle dysfunction in different mouse models of DMD. Study one evaluated the role of Ca2+-activated enzymes (proteases) that activate protein degradation in excitation-contraction (E-C) coupling failure following repeated contractions in mdx and dystrophin-utrophin null (mdx/utr-/-) mice. Single muscle fibers from mdx/utr-/- mice had greater E-C coupling failure following repeated contractions compared to fibers from mdx mice. Moreover, protease inhibition during these contractions was sufficient to attenuate E-C coupling failure in muscle fibers from both mdx and mdx/utr-/- mice. Study two evaluated the effects of overexpressing the Ca2+ buffering protein sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) in skeletal muscles from mdx and mdx/utr-/- mice. Overall, SERCA1 overexpression decreased muscle damage and protected the muscle from contraction-induced injury in mdx and mdx/utr-/- mice. In study three, the cellular mechanisms underlying the beneficial effects of SERCA1 overexpression in mdx and mdx/utr-/- mice were investigated. SERCA1 overexpression attenuated calpain activation in mdx muscle only, while partially attenuating the degradation of the calpain target desmin in mdx/utr-/- mice. Additionally, SERCA1 overexpression decreased the SERCA-inhibitory protein sarcolipin in mdx muscle but did not alter levels of Ca2+ regulatory proteins (parvalbumin and calsequestrin) in either dystrophic model. Lastly, SERCA1 overexpression blunted the increase in endoplasmic reticulum stress markers Grp78/BiP in mdx mice and C/EBP homologous protein (CHOP) in mdx and mdx/utr-/- mice. Overall, findings from the studies presented in this dissertation provide new insight into the role of Ca2+ in muscle dysfunction and damage in different dystrophic mouse models. Further, these findings support the overall strategy for improving intracellular Ca2+ control for the development of novel therapies for DMD.

Applying Mathematical Models to Study the Role of the Immune System in Chronic Myelogenous Leukemia

Although tyrosine kinase inhibitors (TKIs) such as imatinib have transformed chronic myelogenous leukemia (CML) into a chronic condition, these therapies are not curative in the majority of cases. Most patients must continue TKI therapy indefinitely, a requirement that is both expensive and that compromises a patient's quality of life. While TKIs are known to reduce leukemic cells' proliferative capacity and to induce apoptosis, their effects on leukemic stem cells, the immune system, and the microenvironment are not fully understood. A more complete understanding of their global therapeutic effects would help us to identify any limitations of TKI monotherapy and to address these issues through novel combination therapies. Mathematical models are a complementary tool to experimental and clinical data that can provide valuable insights into the underlying mechanisms of TKI therapy. Previous modeling efforts have focused on CML patients who show biphasic and triphasic exponential declines in BCR-ABL ratio during therapy. However, our patient data indicates that many patients treated with TKIs show fluctuations in BCR-ABL ratio yet are able to achieve durable remissions. To investigate these fluctuations, we construct a mathematical model that integrates CML with a patient's autologous immune response to the disease. In our model, we define an immune window, which is an intermediate range of leukemic concentrations that lead to an effective immune response against CML. While small leukemic concentrations provide insufficient stimulus, large leukemic concentrations actively suppress a patient's immune system, thus limiting it's ability to respond. Our patient data and modeling results suggest that at diagnosis, a patient's high leukemic concentration is able to suppress their immune system. TKI therapy drives the leukemic population into the immune window, allowing the patient's immune cells to expand and eventually mount an efficient response against the residual CML. This response drives the leukemic population below the immune window, causing the immune population to contract and allowing the leukemia to partially recover. The leukemia eventually reenters the immune window, thus stimulating a sequence of weaker immune responses as the two populations approach equilibrium. We hypothesize that a patient's autologous immune response to CML may explain the fluctuations in BCR-ABL ratio that are regularly seen during TKI therapy. These fluctuations may serve as a signature of a patient's individual immune response to CML. By applying our modeling framework to patient data, we are able to construct an immune profile that can then be used to propose patient-specific combination therapies aimed at further reducing a patient's leukemic burden. Our characterization of a patient's anti-leukemia immune response may be especially valuable in the study of drug resistance, treatment cessation, and combination therapy.

Development and Application of Predictive Models for Survival, Growth, and Death of Enteric Pathogens in Leafy Greens Supply Chain

Leafy greens are essential part of a healthy diet. Because of their health benefits, production and consumption of leafy greens has increased considerably in the U.S. in the last few decades. However, leafy greens are also associated with a large number of foodborne disease outbreaks in the last few years. The overall goal of this dissertation was to use the current knowledge of predictive models and available data to understand the growth, survival, and death of enteric pathogens in leafy greens at pre- and post-harvest levels. Temperature plays a major role in the growth and death of bacteria in foods. A growth-death model was developed for Salmonella and Listeria monocytogenes in leafy greens for varying temperature conditions typically encountered during supply chain. The developed growth-death models were validated using experimental dynamic time-temperature profiles available in the literature. Furthermore, these growth-death models for Salmonella and Listeria monocytogenes and a similar model for E. coli O157:H7 were used to predict the growth of these pathogens in leafy greens during transportation without temperature control. Refrigeration of leafy greens meets the purposes of increasing their shelf-life and mitigating the bacterial growth, but at the same time, storage of foods at lower temperature increases the storage cost. Nonlinear programming was used to optimize the storage temperature of leafy greens during supply chain while minimizing the storage cost and maintaining the desired levels of sensory quality and microbial safety. Most of the outbreaks associated with consumption of leafy greens contaminated with E. coli O157:H7 have occurred during July-November in the U.S. A dynamic system model consisting of subsystems and inputs (soil, irrigation, cattle, wildlife, and rainfall) simulating a farm in a major leafy greens producing area in California was developed. The model was simulated incorporating the events of planting, irrigation, harvesting, ground preparation for the new crop, contamination of soil and plants, and survival of E. coli O157:H7. The predictions of this system model are in agreement with the seasonality of outbreaks. This dissertation utilized the growth, survival, and death models of enteric pathogens in leafy greens during production and supply chain.

LUNG CANCER IN NEPAL: THE ROLE OF TRADITIONAL TOBACCO PRODUCTS AND COMBUSTION RELATED HOUSEHOLD AIR POLLUTION

The burden of chronic diseases such as cancer is increasing in low and middle income countries around the globe. Nepal, one of the world’s poorest countries, is no exception to this trend, with lung cancer as the leading causes of cancer deaths. Despite this, limited data is available on the environmental and behavioral risk factors that contribute to the lung cancer etiology in Nepal. The objectives of this dissertation are to: 1) investigate the ethnic differences in consumption of local tobacco products and their role in lung cancer risk in Nepal; 2) evaluate urinary metabolite of 1,3-butadiene as a biomarker of exposure to combustion related household air pollution (CRHAP); 3) investigate the association between CRHAP exposure and lung cancer risk using urinary metabolite of 1,3-butadiene as a biomarker of exposure; 4) investigate the association between CRHAP exposure and lung cancer risk using questionnaire based measure of exposure. Lung cancer cases (n=606) and frequency matched controls (N=606) were recruited from B.P. Koirala Memorial Cancer Hospital. We obtained biological samples and information on lifestyles including cooking habits and type of fuels used. We used liquid chromatograph tandem mass spectrometer (LC-MS/MS) to quantify urinary metabolites of 1,3-butadiene in urine samples. We employed a combination of logistic and linear regression models to detect any exposure-disease associations while controlling for known confounding variables. Overall, we found that ethnic groups in Nepal use different tobacco products that have different differing cancer potency -we observed the highest odds ratios for the traditional tobacco products. The biomarker analysis showed strong evidence that monohydroxybutyl mercapturic acid is associated with biomass fuel use among participants. However, we did not find significant association between urinary MHMBA and lung cancer risk. When we used questionnaire based measure of exposure to household air pollution, we observed significant, dose-response associations between CRHAP exposure and lung cancer risk, particularly among never-smokers. Our results show that important role of local tobacco products in lung cancer risk in Nepal. Furthermore, we demonstrate that CRHAP exposure is a risk factor for lung cancer risk, independent of tobacco smoking.

The Role of Attachment in a Social Cognitive Model of Social Domain Satisfaction in College Students

The study examined a modified social cognitive model of domain satisfaction (Lent, 2004). In addition to social cognitive variables and trait positive affect, the model included two aspects of adult attachment, attachment anxiety and avoidance. The study extended recent research on well-being and satisfaction in academic, work, and social domains. The adjusted model was tested in a sample of 454 college students, in order to determine the role of adult attachment variables in explaining social satisfaction, above and beyond the direct and indirect effects of trait positive affect. Confirmatory factor analysis found support for 8 correlated factors in the modified model: social domain satisfaction, positive affect, attachment avoidance, attachment anxiety, social support, social self-efficacy, social outcome expectations, and social goal progress. Three alternative structural models were tested to account for the ways in which attachment anxiety and attachment avoidance might relate to social satisfaction. Results of model testing provided support for a model in which attachment avoidance produced only an indirect path to social satisfaction via self-efficacy and social support. Positive affect, avoidance, social support, social self-efficacy, and goal progress each produced significant direct or indirect paths to social domain satisfaction, though attachment anxiety and social outcome expectations did not contribute to the predictive model. Implications of the findings regarding the modified social cognitive model of social domain satisfaction were discussed.

COLLEGE STUDENTS’ ATTITUDES TOWARD SEEKING PROFESSIONAL HELP: THE ROLE OF SOCIAL CLASS, CLASSISM, AND STIGMA

Research on attitudes toward seeking professional help among college students has examined the influence of social class and stigma. This study tested 4 theoretically and empirically derived structural equation models of college students’ attitudes toward seeking counseling with a sample of 2230 incoming university students. The models represented competing hypotheses regarding the manners in which objective social class, subjective social class, classism, public stigma, stigma by close others, and self-stigma related to attitudes toward seeking professional help. Findings supported the social class direct and indirect effects model, as well as the notion that classism and stigma domains could explain the indirect relationships between social class and attitudes. Study limitations, future directions for research, and implications for counseling are discussed.

The role of the Indian Ocean sector and sea surface salinity for prediction of the coupled Indo-Pacific system

The purpose of this dissertation is to evaluate the potential downstream influence of the Indian Ocean (IO) on El Niño/Southern Oscillation (ENSO) forecasts through the oceanic pathway of the Indonesian Throughflow (ITF), atmospheric teleconnections between the IO and Pacific, and assimilation of IO observations. Also the impact of sea surface salinity (SSS) in the Indo-Pacific region is assessed to try to address known problems with operational coupled model precipitation forecasts. The ITF normally drains warm fresh water from the Pacific reducing the mixed layer depths (MLD). A shallower MLD amplifies large-scale oceanic Kelvin/Rossby waves thus giving ~10% larger response and more realistic ENSO sea surface temperature (SST) variability compared to observed when the ITF is open. In order to isolate the impact of the IO sector atmospheric teleconnections to ENSO, experiments are contrasted that selectively couple/decouple the interannual forcing in the IO. The interannual variability of IO SST forcing is responsible for 3 month lagged widespread downwelling in the Pacific, assisted by off-equatorial curl, leading to warmer NINO3 SST anomaly and improved ENSO validation (significant from 3-9 months). Isolating the impact of observations in the IO sector using regional assimilation identifies large-scale warming in the IO that acts to intensify the easterlies of the Walker circulation and increases pervasive upwelling across the Pacific, cooling the eastern Pacific, and improving ENSO validation (r ~ 0.05, RMS~0.08C). Lastly, the positive impact of more accurate fresh water forcing is demonstrated to address inadequate precipitation forecasts in operational coupled models. Aquarius SSS assimilation improves the mixed layer density and enhances mixing, setting off upwelling that eventually cools the eastern Pacific after 6 months, counteracting the pervasive warming of most coupled models and significantly improving ENSO validation from 5-11 months. In summary, the ITF oceanic pathway, the atmospheric teleconnection, the impact of observations in the IO, and improved Indo-Pacific SSS are all responsible for ENSO forecast improvements, and so each aspect of this study contributes to a better overall understanding of ENSO. Therefore, the upstream influence of the IO should be thought of as integral to the functioning of ENSO phenomenon.