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Immune self-tolerance, the absence of an immune response against the body, is an active process involving several complementary mechanisms. The autoimmune regulator gene (Aire) plays a critical role in this process by inducing the expression of a diverse set of tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs). These TSAs are presented and help eliminate or control self-reactive thymocytes. The expression of Aire is highly restricted, occurring in adults only in mTECs and extrathymic Aire-expressing cells (eTACs), which are found in secondary lymphoid tissues. How this specific pattern of expression is achieved has remained unclear. Although RANK and non-canonical NF-κB signaling are known to be important for the development and maintenance of Aire-expressing mTECs, the cis-regulation of Aire is poorly understood. We aimed to discover distal cis-regulatory elements that control Aire. Here, we identify a conserved DNA element that is essential for Aire expression. We find enrichment of enhancer-associated histone marks near this element, which we call ACNS1 and show to be NF-κB-responsive. We demonstrate that a 4.3-kb region, which includes ACNS1 and the Aire promoter, is sufficient to recapitulate the cell-type specificity of Aire expression. We show that ACNS1 is essential for Aire expression in mTECs and eTACs in vivo and is required to prevent spontaneous autoimmunity. We also observe that Aire and Dnmt3l are co-expressed and that ACNS1 is necessary for thymic Dnmt3l expression. Finally, we present preliminary evidence for additional Aire cis-regulatory elements. Together, these findings further our understanding of the control of immune tolerance and may lead to improvements in the diagnosis and treatment of autoimmune disease.

The mammary gland is a branched, secretory organ that that is composed of diverse cells types. The populations that comprise the mammary epithelium include mammary stem cells, lineage-restricted progenitors, and differentiated luminal and basal cells. Breast cancer develops when one or a subset of these epithelial cells undergoes transformation, resulting in disease that can vary broadly in pathology and prognosis. For example, gene expression analysis of a large cohort of breast cancers has classified tumors into distinct subtypes, including: HER2-positive, Basal-Like, Luminal A, and Luminal B. Furthermore, hormone receptor status is also used in the clinic to classify tumors and define the course of treatment. Factors that may contribute to this broad breast cancer diversity are still unclear; however, we hypothesize that breast cancer phenotype may be influenced by intrinsic properties of the originating cell. Therefore, to determine whether specific cells types influence breast cancer pathology, the goal of this dissertation has been to develop a series of mouse models of breast cancer that can be used to target oncogene expression to distinct mammary epithelial cell (MEC) lineages. One such model was generated by infecting mouse MECs with a lentivirus expressing the polyoma virus middle T antigen oncogene, then cells were enriched for specific cell lineages using fluorescence activated cell sorting, and were orthotopically transplanted to generate tumors. Resulting tumors were classified by histology, estrogen receptor status, molecular subtype, and metastatic propensity. Our results demonstrated that each MEC population we evaluated could give rise to diverse tumor types; however, we also determined that MEC populations exhibited differences in their propensity for tumor histology, molecular subtype, and metastatic potential. Furthermore, we developed an additional approach of targeting specific gene expression to distinct MEC populations. In this system, lentiviral constructs were designed to express a specific gene after Cre-mediated recombination. Then, MECs isolated from mice that express Cre iv through cell lineage-restricted promoters were infected with the lentivirus and orthotopically transplanted or analyzed by three-dimensional culture. We validated this approach by targeting fluorescent Tomato reporter and polyoma virus middle T antigen oncogene expression to specific MEC populations in vitro and in vivo.

The maintenance of metabolic homeostasis presents a major challenge to animal biology. Dysregulation of this process is central to the etiology of diabetes and obesity, which have placed an ever-increasing burden on global human health. Nuclear receptors are a family of ligand-regulated transcription factors that play important roles in preventing metabolic dysfunction by coordinating downstream transcriptional programs to support physiological homeostasis. This thesis is focused on one of these nuclear receptors, Hepatocyte Nuclear Factor 4A (HNF4A), which acts as a critical regulator of systemic glucose homeostasis. Mutations in HNF4A were established 20 years ago as the monogenic cause of an inherited form of diabetes called Maturity-Onset Diabetes of the Young 1 (MODY1). The mechanistic link between HNF4A dysfunction and diabetes onset, however, remains unclear. In this dissertation, I present work that provides new insights into the mechanisms by which HNF4 supports physiological homeostasis and prevents diabetes. The fruit fly Drosophila melanogaster provides an ideal system for studies of metabolism and physiology that can guide our understanding of these pathways in mammals. We developed and optimized methods for studies of metabolism in Drosophila to further advance its utility in these efforts. Using these techniques, I investigated potentially conserved roles for Drosophila HNF4 (dHNF4) in supporting development and metabolic homeostasis. Through this work, I discovered that dHNF4 mutants recapitulate hallmark features of MODY1, including hyperglycemia and impaired glucose-stimulated insulin secretion (GSIS). dHNF4 functions in the adult insulin producing cells and the fat body to support GSIS and peripheral glucose clearance during adulthood. These functions are linked to a role for dHNF4 in promoting mitochondrial activity in the mature animal through up-regulation of both nuclear and mitochondrial-encoded genes involved in oxidative phosphorylation (OXPHOS). By extending my analysis to mammals through a collaborative effort with several labs, we uncovered evidence suggesting that the role for HNF4 in supporting mitochondrial function has been conserved through evolution. Taken together, my work has identified HNF4 as an important regulator of mitochondrial activity, and suggests that this function has been conserved through evolution to support normal development, physiology, and metabolic homeostasis.

Childhood obesity is a major focus of public health. The high rates of childhood obesity can be partially attributed to the increased availability and consumption of energy-dense and nutrient-poor foods and excess time spent in sedentary behavior. The purpose of this study was to determine whether after-school physical activity and dietary habits predict cardio-metabolic risk in a sample of ethnic minority elementary-school-aged children from low-income schools. Participants were a convenience sample of 92 children (3rd-6th grades) recruited from four Title I schools located in a metropolitan area from the Mountain West region of the United States. Children completed portions of the After School Student Questionnaire (ASSQ) and Physical Activity Questionnaire for Children (PAQ-C) to measure after-school physical activity and nutrition. Blood pressure, waist circumference, and cardio-metabolic blood markers were collected in a fasted state before school hours to calculate a continuous metabolic syndrome (MetS) composite score. Predictive relationships were analyzed using a multiple linear regression model. Neither physical activity nor nutrition were predictive of a MetS score. However, there was a linear, positive, and moderate correlation between physical activity and nutrition scores (r = 0.29, p < 0.05). In a secondary analysis, a linear regression established the relationship between after-school physical activity and nutrition. The linear model indicated that a one-unit increase in PA score was associated with 1.28-unit increase in nutrition score. The findings indicate that cardio-metabolic health cannot be predicted based on self-reported after-school physical activity and nutrition habits; however, self-reported PA and nutrition habits are linearly related. Overall, the majority of children in this study exhibited favorable cardio-metabolic health. In fact, 84.8% of the subjects had two or fewer of the risk factors for metabolic syndrome and 40.2% of subjects displayed none of the risk factors. Further research is needed in determining whether after-school physical activity and nutrition habits influence cardio-metabolic health.

Accurate genotyping of the human leukocyte antigens (HLA) are crucial for the success of hematopoietic stem cell and solid organ transplantation. Over the past 50 years, numerous methodologies have been used for HLA typing but technological limitations have prevented full interrogation of the HLA gene thus resulting in allelic ambiguity. In order to resolve these ambiguities, additional testing is often required, leading to increased expense and delay in reporting the genotyping results. Recently, advances in nucleic acid sequencing technologies, generally referred to as next-generation sequencing (NGS), have become available. HLA genotyping by NGS is poised to become the new gold standard for several reasons. First, the entire gene can be interrogated, as opposed to a few exons, thus enabling greater resolution of known polymorphisms outside of the T cell recognition site on the HLA molecule. Secondly, missing genetic sequences in the curated databases will become more complete thus improving the algorithms used for alignment to a reference sequence. Lastly, NGS will become the new gold standard because high-resolution genotyping can be achieved without the need for additional time and laboratory resources in order to resolve allelic ambiguities and meet typing requirements required by regulatory agencies. In this thesis I discuss my efforts to employ this new technology by developing an assay for genotyping of the HLA-A, B, C, DRB1, and DQB1 genes. In the new assay, 98% of all alleles typed correctly and unambiguously without the need for any secondary testing. This in-house method is later compared with two recently released commercial kits and 100% concordance was found between the three methods. Differences in workflow are compared and contrasted. In conclusion, we show that HLA genotyping by NGS produces more correct and unambiguous results than traditional Sanger sequencing without the need for reflex testing.

This dissertation presents original research that improves the ability of magnetic resonance imaging (MRI) to measure temperature in aqueous tissue using the proton resonance frequency (PRF) shift and T1 measurements in fat tissue in order to monitor focused ultrasound (FUS) treatments. The inherent errors involved in measuring the longitudinal relaxation time T1 using the variable flip angle method with a two-dimensional (2D) acquisition are presented. The edges of the slice profile can contribute a significant amount of signal for large flip angles at steady state, which causes significant errors in the T1 estimate. Only a narrow range of flip angle combinations provided accurate T1 estimates. Respiration motion causes phase artifacts, which lead to errors when measuring temperature changes using the PRF method. A respiration correction method for 3D imaging temperature of the breast is presented. Free induction decay (FID) navigators were used to measure and correct phase offsets induced by respiration. The precision of PRF temperature measurements within the breast was improved by an average factor of 2.1 with final temperature precision of approximately 1 °C. Locating the position of the ultrasound focus in MR coordinates of an ultrasound transducer with multiple degrees of freedom can be difficult. A rapid method for predicting the position using 3 tracker coils with a special MRI pulse iv sequence is presented. The Euclidean transformation of the coil's current positions to their calibration positions was used to predict the current focus position. The focus position was predicted to within approximately 2.1 mm in less than 1 s. MRI typically has tradeoffs between imaging field of view and spatial and temporal resolution. A method for acquiring a large field of view with high spatial and temporal resolution is presented. This method used a multiecho pseudo-golden angle stack of stars imaging sequence to acquire the large field of view with high spatial resolution and k-space weighted image contrast (KWIC) to increase the temporal resolution. The pseudo-golden angle allowed for removal of artifacts introduced by the KWIC reconstruction algorithm. The multiple echoes allowed for high readout bandwidth to reduce blurring due to off resonance and chemical shift as well as provide separate water/fat images, estimates of the initial signal magnitude M(0), T2 * time constant, and combination of echo phases. The combined echo phases provided significant improvement to the PRF temperature precision, and ranged from ~0.3-1.0 °C within human breast. M(0) and T2 * values can possibly be used as a measure of temperature in fat.

Fetal exposure to maternal tobacco smoke (MTS) causes growth restriction and alters lung development in humans and animal models. Lung development depends upon molecular mechanisms including PPARγ and Wnt signaling. Recent reports suggest that chromatin-mediated negative cross-talk between PPARγ and Wnt signaling reduces transcription of Wnt output genes. This chromatin-mediated cross-talk is initiated by the lysine histone methyltransferase, and PPARγ target gene, Setd8. We previously showed that lung protein abundance of PPARγ is decreased by MTS exposure in both male and female rat pups. However, the effect of MTS exposure on Setd8 abundance, Wnt signaling, and expression of Wnt target genes in the rat lung is unknown. We hypothesize that MTS exposure decreases Setd8 protein abundance in newborn rat lung. We also hypothesize that MTS exposure decreases transcription of Wnt output genes, in the context of increased Wnt signaling, in newborn rat lung. Pregnant rats were exposed to room air (Control) or tobacco smoke (MTS) from gestational day 11.5 to term (day 22.5). Newborn pups were euthanized and lung tissue collected. We used western blot to measure β-catenin and Setd8 protein abundance in newborn female and male rat pup lung. We used real-time RT-PCR to measure mRNA transcript levels of Lef1, Tcf4, Axin2, MMP-9, and MMP-12 in newborn female and male rat lung. Setd8 protein abundance did not change in MTS exposed female rat lung and was significantly decreased in MTS exposed male rat lung compared to sex-matched controls. β-catenin protein abundance was increased in both female and male MTS exposed rat lung compared to sex-matched controls. mRNA transcript levels of Wnt transducer Lef1 were unchanged in MTS exposed female and male newborn rat lung compared to sex- matched controls. Tcf4 mRNA transcript levels significantly increased in MTS exposed female and male newborn rat pup lung compared to sex-matched controls. Axin2 mRNA transcript levels significantly decreased in only female newborn rat lung, while male Axin2 mRNA transcript levels were unchanged compared to sex-matched controls. mRNA transcript levels of Wnt target gene MMP-9 were significantly decreased in MTS exposed female and male newborn rat lung compared to sex-matched controls. MMP-12 mRNA transcript levels were increased in both female and male newborn rat lung compared with sex-matched controls. Our results demonstrated that MTS exposure causes changes in Setd8 protein abundance as well as mRNA transcript levels of Wnt transducers and target genes in newborn rat lung. We speculate that the interaction of MTS exposure, the PPARγ-Setd8- H4K20me1 pathway, and Wnt signaling pathway contribute to these changes.

Magnetic resonance imaging (MRI) is a popular imaging modality that allows for noninvasive detection of diseases without the use of ionizing radiation. Every imaging modality comes with its set of challenges and limitations. Since the rate at which data can be collected using an MRI scanner is relatively slow, data undersampling is often employed in order to accelerate acquisitions. This undersampling leads to artifacts in the reconstructed image due to the lack of sufficient data. In this dissertation, novel reconstruction techniques are developed to reconstruct high-quality and artifact-free images using a compressed sensing (CS) formulation. The reconstruction techniques are specifically designed and developed to handle the various challenges that occur in acquiring high-resolution myocardial perfusion images. Since the reconstruction of these images is often time consuming, techniques are developed to allow rapid reconstruction of images. Rapid reconstruction aids in the transition of CS techniques for myocardial perfusion MRI into a tool that is routinely used by clinicians.

BALB/c mice have been used to study multiple diseases that cause inflammation such as Lyme arthritis, rheumatoid arthritis, and systemic lupus erythematosus. Lyme disease or, more specifically, Lyme arthritis is caused by Borrelia burgdorferi that has disseminated into joint tissues. A spectrum of Lyme disease severity and symptoms has been seen in Lyme disease patients, which suggests that genetics play a role in host response. Inbred mouse strains display the range of arthritis severity seen in patients from the mild arthritis phenotype in C57BL/6 mice to the severe arthritis phenotype in C3H mice. The C3H mouse model develops severe Lyme arthritis that can be attributed to 2 important inflammatory factors: a hypomorphic allele of GusB and hyper production of Type I IFN-induced transcripts. Using SNP-based assessment, the BALB/c mouse was found to be closely related to the C3H mouse; however, the BALB/c mouse does not have the hypomorphic GusB allele. It was hypothesized that the BALB/c mouse, which develops severe, dose-dependent arthritis, would also hyperproduce Type I IFN-related transcripts. This study found that the BALB/c mouse does not develop an IFN profile similar to the C3H mouse. Multiple inflammatory markers were analyzed to compare and contrast BALB/c and C3H mice, and TNFα was determined to be elevated in the BALB/c mouse above the levels in the C3H mouse both in vivo during infection and in vitro using bone marrow-derived macrophages. This suggests an important role of the innate immune response in these mice. Interestingly, peritoneal macrophages derived from BALB/c were able to internalize B. burgdorferi much better than C3H-derived peritoneal macrophages. This suggests an elevated intrinsic response in controlling B. burgdorferi numbers. TNFα blockade has been shown to be beneficial in relieving symptoms in rheumatoid arthritis patients, so it was hypothesized that TNFα blockade in BALB/c mice would reduce Lyme arthritis symptoms. This study did not see any benefit to TNFα blockade in Lyme arthritis reduction. Overall, this study has shown that there are still mechanisms that are yet to be fully understood in the pathogenic relationship between the BALB/c mouse and the spirochete bacteria B. burgdorferi.

Many studies have estimated the treatment effect of different antiretroviral therapies (ARTs) on human immunodeficiency virus (HIV) patients’ virologic/immunologic outcomes. However, evidence is lacking on how adherence to ARTs influences treatment effe

In the field of ecology, regime shifts are often abrupt and catastrophic occurrences with far reaching effects on global and local ecosystems which result in distinct changes in the composition and function of an ecosystem. The boreal forest provides a lo

Background: Larger dishware sizes have been shown to increase food portion sizes served and consumed by adults and children. Less is known about whether dishware size and design influences the amount of food caregivers serve to children. Whether smaller portion design plates can be used to help parents serve age-appropriate portion sizes to their children is unknown. This research is the first to examine the effects of dishware size and design on portion sizes served by caregivers. Methods: A within-subjects quasi-experimental design was used to determine the effects of dishware size and design on the portion sizes served by low-income caregivers to their preschool aged children during a buffet style meal. Three plate conditions were compared: a portion design plate, a plain child size plate, and an adult size plate. We hypothesized that when using the portion design plate, caregivers would serve the smallest amount of food compared to when using either the plain child size plate or the adult size plate. The amount of food served by caregivers to children was assessed using weighted methods. Results: The total amount of food (g) and energy (kcal) served did not differ across dishware conditions. Conclusions: Dishware size and design did not influence portion sizes served by caregivers. Additional factors may affect caregiver serving behaviors.

Bone morphogenetic proteins (BMPs) drive a range of cellular processes especially in the early stages of embryonic development. This family of proteins acts as one of the most important extracellular signals in development pattern formation across the animal kingdom. Cells in embryos differentiate into different cell types in response to the concentration level of BMP. This complex process is regulated by multiple regulators that serve to tune the signal response. Extensive experimental and computational research has been performed to analyze BMP regulation in Drosophila, a widely studied model organism, and has advanced our understanding of animal development. Because of BMP’s role in regulating cell growth and differentiation, identifying key players and their dynamic regulatory interactions in BMP gradient formation provides useful information to understand how BMP signaling works across the animal kingdom, and thus lead to potential treatment of related diseases and other clinical applications. Although extensive experimental study and system biology approaches like mathematical modeling have been done to investigate BMP regulations, there are still many unanswered questions on the functions of specific BMP regulators. Specifically, in this study we aim to investigate the roles of two feedback regulators, Crossveinless-2 (Cv2) and Eiger (Egr). These two proteins cooperate with extracellular modulators like short gastrulation (Sog), and guide the formation of BMP signal gradient in Drosophila embryonic development. A genetic network containing Eiger which promotes BMP signaling and Cv2 antagonizing BMP signaling was identified by Gavin-Smyth et al. via experimental approaches. In this report, mathematical models were built to discover the mechanism of BMP regulation with these potential feedback loops. In order to explore Cv-2 and Eiger's roles in formation of BMP signal gradient, in this study, we developed a mathematical model with BMPs, the intracellular messenger pMad and regulators including Eiger and Cv2 for identifying potential roles of Cv2 and Eiger. A one-dimensional spatial model representing the cross-section of a Drosophila embryo was built. In order to compare models with multiple potential proposed mechanisms, we collected and processed data from experimental research and tested six alternative models with different proposed functions of Eiger and Cv2. Then these models were compared using Pareto frontiers, a widely-used concept in multi-objective optimization. While comparing different models, we developed a comparatively easy, yet efficient way of finding the Pareto fronts. The results support a mechanism where i) Eiger is potentially preventing BMP releasing after binding to its receptor; ii) Cv2 is intermediating BMP by forming a complex with both BMP and its receptor, thus having a potential biphasic effect on BMP signaling, similar to Cv2 in Drosophila wing disc. This finding could provide new insight into the function of Eiger and Cv2, and potentially proper directions of further study.

In the United States, the emergence of an outpatient-centered, drug-based model of mental health care was physically feasible from the 1950s onward, with the introduction of thorazine and other first-generation antipsychotics. However, it was not until 1981, with President Reagan’s veto of the Mental Health Systems Act, that American mental health policy tipped over definitively into the outpatient-centered, drug-based model. In this quantitative study of the formation of policy preference, the delay between the feasibility of the outpatient-centered, drug-based model and its adoption was explored through five research questions answered through corpus analysis and time series statistics: How do shifts in (1) the sentiments of the American public; (2) the opinions of psychiatrists, psychologists, and other mental health experts; (3) formal and informal lobbying efforts by pharmacological companies and other commercial stakeholders in mental health; (4) the policy of individual states; and (5) the policy of the federal government explain the delay between the appearance of thorazine and the adoption of the current model of American mental health care? These questions were answered through techniques such as Chow breakpoint analysis, Markov switch models, vector auto-regression (VAR), and autoregressive distributed lag (ARDL) models. It was found that both the outpatient-centered, drug-based model of mental health and community mental health centers viewed for popularity with the public, Congress, and mental health professionals for several years, thus delaying a transmission of a firm preference for the outpatient-centered, drug based model of mental health from the public to Congress. This finding was explored through the theories of multiple streams and disjointed incrementalism. The study demonstrated the existence of a robustly democratic period of policy articulation and explanation followed by a transference of public preference into governmental preference.

Xue, Pengcheng. Ph.D., Purdue University, December 2016. The Relationship between Protein and Phosphorus Digestion and Retention in Growing Pigs and Broiler Chickens. Major Professor: Dr. Layi Adeola. The objective of this study was to investigate the relationship between protein and P digestion and retention in growing pigs and broiler chickens. The methodology of determining the digestibility of AA and P and the effect of dietary N and P on the digestion and retention of these two nutrients were investigated. An experiment was conducted in growing pigs to investigate the additivity of AID or SID of CP and AA in mixed diets containing multiple protein sources. Using the determined AID or SID for CP and AA in corn, SBM, DDGS, and canola meal, the AID or SID for 4 mixed diets was predicted and compared with determined AID or SID, respectively. Eighteen growing pigs (initial BW = 61.3 ± 5.5 kg) with surgically fitted T-cannula were assigned to a duplicated 9 × 4 incomplete Latin square design with 9 diets and 4 periods. A nitrogen-free diet was included to estimate basal ileal endogenous loss of AA; 4 semi-purified diets to determine the AID and SID of CP and AA in the 4 ingredients; and 4 mixed diets to test the additivity of AID and SID. The results substantiate the notion that SID of AA are more accurate than AID for predicting ileal digestibility of AA in mixed diets containing multiple protein sources. To determine the TTTD of P in triticale DDGS for growing pigs with or without phytase using the regression method, six diets were formulated in a 3 × 2 factorial arrangement, including 3 levels of triticale DDGS (300, 400, or 500 g/kg) and phytase (0 or 500 FTU/kg of diet). A total of 48 barrows (initial BW 22.2 ± 1.3 kg) were assigned to the 6 diets in a randomized complete block design. There was a 5-d adjustment period followed by a 5-d total collection of feces. The results indicated that phytase improved ATTD of P in triticale DDGS (P < 0.001). In diets without added phytase, the ATTD of P in triticale DDGS was 65.0, 67.7, and 63.2% for the diets with 300, 400, and 500 g/kg triticale DDGS, respectively; the corresponding values for diets with added phytase were 77.3, 76.3, and 75.7%.The TTTD of P was estimated at 75.4% for triticale DDGS or 81.1% with added phytase, respectively. The difference between the TTTD with or without phytase was not statistically significant. For triticale DDGS, the supplementation of 500 FTU/kg phytase in the diet could increase the ATTD of P (P < 0.001), but not the TTTD of P. The effect of dietary CP concentrations on ileal P digestion in growing pigs was investigated in the third study. A total of 18 ileal-cannulated pigs (initial BW 44.2 ± 3.2 kg) were used in a duplicated 9 × 3 incomplete Latin Square design, with 9 treatments and three 7-d experimental periods giving 6 replicates per treatment. The 9 treatments consisted of 1 nitrogen-free diet to estimate basal endogenous loss of AA, and 8 corn-soybean meal-based diets in a 2 × 4 factorial arrangement, which included 2 CP concentrations (6.9 or 13.4%) and 4 ATTDP concentrations (0.09, 0.16, 0.24, or 0.32%). Low CP diets limited ileal digested P (g/kg•DMI) (P < 0.05). The ileal digested P (g/kg•DMI) increased linearly (P < 0.01) with increasing ATTDP concentrations in the low CP group, but the pattern was linear (P < 0.01) and quadratic (P < 0.01) in the high CP group. In the low and high CP diets, the determined true ileal digestibility of P in mono-calcium phosphate was 54.4% and 75.6%, respectively. In conclusion, this research indicated that the ileal digestion of P could be limited by protein deficiency. Thus dietary CP concentration should be considered in P digestibility related studies. The quantitative relationship between N and P digestion and retention in broiler chickens and growing pigs was determined in the last two studies. In the broiler chicken study, a total of 384 14-d-old male broiler chickens were used in a randomized complete block design with 8 treatments and 6 replicates per treatment in a 7-d experimental period. There were 8 corn-soybean meal-based diets in a 2 × 4 factorial arrangement, which included 2 CP concentrations (10.7 or 21.5%) and 4 ATTDP concentrations (0.18, 0.32, 0.45, or 0.59%). Results showed that low dietary CP concentration limited growth performance (P < 0.01), pre-cecal digestion and total tract retention of P (P < 0.01), and NaPi-IIb gene expression ( P < 0.05). Pre-cecal digestion and total tract retention of P (g/kg DM intake) linearly increased (P < 0.01) with increasing ATTDP concentrations in both low and high CP groups. In conclusion, this study suggests an interrelationship between N and P digestion such that CP deficiency decreased the growth performance of birds consequently reducing pre-cecal P digestion in broiler chickens. Total tract retention of CP and P are linked with each other and body tissue growth may be a driver of the deposition of these two nutrients. In the growing pig study, a total of 72 growing pigs (initial BW 20.9 ± 0.8 kg) were used in a randomized complete blocked design, with 9 treatments and four 10-d experimental periods giving 8 replicates per treatment. The pigs were blocked by BW and allotted to 9 treatments with a 3 × 3 factorial arrangement consisting of 3 CP concentrations (5.5, 9.7, or 13.9%) and 3 ATTDP concentrations (0.11, 0.19, or 0.27%). The determined TTTD of P in MCP for the 5.5, 9.7, and 13.9% CP diets were 80.5, 82.6, and 87.9%, respectively. There were no statistical differences among the three TTTD estimates. In the nitrogen utilization results, increasing dietary P level decreased the urine nitrogen output (P < 0.05). In conclusion, the results indicated that dietary CP deficiency may limit total tract P digestion and retention. Yet the quantitative relationship between total tract N and P retention remains unclear. In summary, the SID of most AA in corn, SBM, canola meal, and DDGS, are additive in complete diets. The regression method can be used to determine the true P digestibility and retention in feed ingredients for growing pigs and broiler chickens. Dietary CP deficiency can limit ileal and total tract P digestion. In broiler chickens, the retention of P is correlated with the retention of N. Yet in growing pigs this relationship has not been observed. Body weight gain might be the driving factor for the correlation between N and P retention in growing pigs and broiler chickens.

My dissertation research has focused on the development of ambient ionization – mass spectrometry (MS) for clinical measurements, specifically intrasurgical cancer detection. The molecular differences between normal and cancerous tissue were detected via direct tissue analysis in vitro by touch spray ionization (TS) or by analyzing sectioned or smeared tissue using desorption electrospray ionization (DESI). The physical form of the tissue, e.g. in vitro sampling, sectioned, or smeared, was inconsequential in differentiating normal from cancerous tissue; however, the spectra acquired by TS and DESI differed due to differences in ionization processes. We envision that TS-MS and DESI-MS could impact diagnostic medicine, for example in providing surgeons with rapid, near real-time information as to tissue disease state, i.e. normal or tumor. Disease state information provided to surgeons about discrete pathologically ambiguous areas, ideally intrasurgically via TS or DESI-MS smear analysis, could improve the completeness of tumor resection while minimizing unintended damage to adjacent tissue. Touch spray ionization was developed for intrasurgical detection of cancer; TS greatly simplifies MS analysis by using the same device for in vivo sampling and subsequent ionization. Frozen tissue sections were sampled and analyzed by TS-MS providing the ability to differentiate normal from human prostate cancer, via lipid profiles, using multivariate statistics. The next proof-of-concept step for TS-MS was the analysis human kidney cancer specimens in vitro, immediately following resection. TS-MS analysis of untreated kidney tissue emulated intrasurgical use, e.g. the presence and co-sampling of biofluids such as blood. Regardless, normal renal tissue and kidney cancer was differentiated using lipid profiles and multivariate statistics. Desorption electrospray ionization (DESI) - MS imaging of tissue sections differentiated normal from tumor in all cancers studied. DESI-MS imaging of human prostate and human kidney tissue sections were performed to corroborate TS-MS results. Human brain cancer, a major focus of my dissertation research, was studied by imaging tissue sections using DESI-MS to establish the characteristic chemical features, e.g. lipid and metabolite profiles, that distinguish normal brain parenchyma from gliomas and different brain tumors. It was found that information in the negative ion mode lipid profile, positive ion mode lipid profile, and negative ion mode metabolite profile is able to discriminate brain parenchyma (grey and white matter) and gliomas, the most common form of malignant brain tumor. Further, the negative mode lipid and metabolite profiles also proved capable of discriminating different types of brain tumors (gliomas, meningiomas, and pituitary tumors) which account for ~80% of all central nervous system tumors. DESI-MS imaging of effaced or otherwise pathologically ambiguous frozen tissue sections offered the ability to determine the underlying brain parenchyma in cancerous samples – something that traditional morphologic evaluation was not able to determine. Further, DESI-MS was able to detect molecular changes resulting from varying amounts of glioma tumor cells present within infiltrated tissues. The tumor cell percentage of these samples was predicted using N-acetyl-aspartic acid, a neurometabolite which was found to decrease in cancerous tissue, and matched well with histopathologic evaluation. The transition from DESI-MS imaging of sectioned tissue to DESI-MS analysis of tissue smears was driven by the time restriction of intrasurgical application. The potential of DESI-MS analysis of smears was first demonstrated upon canine non-Hodgkin’s lymphoma fine-needle aspirate smears which provided similar sensitivity and specificity values to that of tissue section imaging but is technically less demanding and decreased analysis time. DESI-MS imaging of tissue sections established that MS profiles contained the sufficient information for diagnosis; whereas DESI-MS analysis of tissue smears made the intrasurgical analysis of human brain tumors feasible. The observed lipid or metabolite profiles were not significantly altered by the physical act of smearing and their signal intensities were comparable to those of tissue sections. Further, the chemical information obtained from tissue smears was equivalent to those of tissue sections as determined by canonical component analysis. The culmination of my dissertation research was the creation and implementation of an intrasurgical DESI-MS tissue smear analysis method for human gliomas. Preliminary results from the initial intrasurgical cases analyzed using the developed DESI-MS method are discussed.

The nitrogen (N) and oxygen (O) stable isotope composition (δ15N & δ18O) of nitrogen oxides (NOx )may be a useful tool for constraining NOx emission sources as well as for understanding the atmospheric oxidation pathways responsible for its removal if various NOx sources and sink processes exhibit characteristic isotopic compositions (“fingerprints”). However, this requires (1) an accurate and complete inventory of δ15N(NOx) values from major emission sources, (2) an assessment of the kinetic and equilibrium isotope effects that can impact δ15N and δ18O values of NOx, (3) and test these assumptions by conducting accurate in situ δ15N and δ18O measurements of atmospheric NOx. To this end, I have characterized the δ15N(NOx) signatures from various fossil-fuel NOx sources, including buses, trucks, lawn equipment, natural gasfired boilers, and airplanes. These δ15N(NOx) source characterization studies along with prior studies indicate that soil emission (nitrification/denitrification), “thermal” NOx producedfromfossil-fuelcombustion, and“source” NOx producedfromcoal-fired power plants have relative distinctive values. In addition, both my experimental and theoretical investigations on the isotope effects associated with NOx oxidation indicate that isotopes effects via equilibrium isotope exchange and kinetic isotope effects occurring during NOx oxidation reactions may influence the δ15N and δ18O values of atmospheric nitrate. Using these calculated isotope effects, I developed a simple model for the production of atmospheric nitrate through its three major pathways thatinclude(1)NO2 +•OH→HNO3, (2)N2O5 +surface→2HNO3, and(3)NO3+ R→•R. This model indicated that these pathways result in distinctive δ18O-δ15N relationships that tend to match reported literature values. Finally, in order to evaluate the influences of NOx emission sources and isotope effects on the isotope composition of NO2, which serves as precursor molecule to atmospheric nitrate, ambient NO2 was collected and analyzed for 15N and 18O . These results suggest that δ18O of NO2 has a distinctive diurnal profile reflecting the photochemical cycling of NOx while δ15N of NO2 tends to track with NOx sources with small but significant isotope effects altering daytime δ15N(NO2) by approximately 2-4%. Overall, this research has refined the “fingerprints” of atmospheric NOx and will be useful for future studies aimed at understanding regional and spatial distributions in NOx emission budgets and tracing NOx oxidation chemistry.

Glioblastoma Multiforme GBM is a very aggressive type of malignant brain tumors that affects peoples’ lives. The diffusive, infiltrative, and metastatic behaviour of GBM is the major reason for the disease recurrence. The morphological and immunohistological characteristics of Central Nervous System (CNS) tumors including GBM are heterogeneous. GBM is either primary (de novo) or secondary to low-grade astrocytomas. Current treatment options include surgery, radiotherapy, and temozolomide chemotherapy have not achieved any improvement in success rates over the past decades. The survival time reached by GBM patients was approximately 12 months only after being treated with radiotherapy alone without temozolomide. However, the median survival time has been estimated as 14.6 months in patients who received the combined treatment of radiotherapy and chemotherapy with temozolomide. Temozolomide is an alkylating agent that exhibit antitumor activity and prescribed as a single agent for the treatment of recurrent glioma. It diminishes the O6-methylguanine-DNA methyltransferase (MGMT) enzyme, which is responsible for DNA repair. GBM Patientswith activated O6-alkylguanine transferase AGT enzyme were reported to develop resistant to temozolomide. Targeted therapies are other options for GBM patients. Bevacizumab blocks the effect of Human Vascular Endothelial Growth Factor (VEGF) and inhibits tumor vascular growth. Regulatory concerns about clinical trials conducted with bevacizumab including trial design problems have been raised. Targeting the immune system will identify successful treatments for GBM with significant clinical benefits. The use of active immunotherapy to increase the native immune response or passive immunotherapy to target the tumor cells in GBM patients are under investigation. Dendritic Cells DCs are the most potent antigen presenting APCs in the immune system. DCs have the ability to stimulate the native T cells and induce primary immune responses and peripheral immunological tolerance through capturing, processing neoantigens, which are formed and released by oncogenesis, and presenting the captured antigens on Histocompatibility Complex I and II (MHCI and MHCII) molecules to T cells. After that T cell responses against the cancer-specific antigens are primed and activated. T cells infiltrate the tumor bed, specifically recognize, bind to, and kill their target cancer cell. The sources of antigen that have been used in DC immunotherapy include exogenous MHC-restricted peptides, acid-eluted tumor peptides, tumor RNA and cDNA, viral vectors, apoptotic tumor cells, tumor cell lysate, and whole glioma cells. Clinical trials showed that treating GBM patients with surgery, TMZ, and Dendritic Cell DC vaccines was safe and achieved meaningful clinical outcomes. The purpose of this research is to investigate the clinical outcomes of DC vaccines for the treatment of GBM patients through the published data on the rapidly growing field of clinical trials. Furthermore, this research focuses on investigating immune responses that are related to the most beneficial clinical outcomes to identify new prognostic biomarkers and therapeutic targets. This research’s findings have showed evidence that many different variables were associated with different cancer specific immune responses and clinical outcomes. These variables include loading DC vaccines with tumor material or Tumor Associated Antigens TAAs, and combining DC vaccines with different pre and post vaccination treatment strategies. DC vaccine pulsed with specific synthetic antigens have achieved more beneficial clinical outcome than DC vaccines loaded with tumor material. Pre-vaccination treatment strategy with TMZ has increased the Overall Survival and the Progression Free Survival of GBM patients. Although, pre-vaccination treatment strategy with TMZ+RT has achieved significant improvement of Overall Survival, there was no improvement of the Progression Free Survival associated with the strategy.

Nutrient abatement programs, originally enacted to counter eutrophication have successfully suppressed nutrient loads and relative primary production in marine and freshwater systems. Recently, the additive impacts of invasive filter feeders have further reduced offshore primary production in several aquatic systems throughout the world. It has been hypothesized that the biological activity of these invasive species may sequester nutrients within the nearshore benthic environment, creating steep gradients in primary production between nearshore and offshore habitats. In the Laurentian Great Lakes, past studies of oligotrophication have primarily focused on food web responses in offshore habitats. Organisms at higher trophic levels have been impacted by reduced productivity in offshore waters and may now rely more heavily on nearshore production pathways. This study analyzed long-term changes in surface chlorophyll a concentrations along a nearshore to offshore gradient and the subsequent responses in physiological condition of the secondary consumer Bythotrephes longimanus (hereafter referred to as Bythotrephes). First, we examined long-term (1998-2013) seasonal and regional trends in surface chlorophyll a concentrations in the main basins of lakes Michigan and Huron using satellite imagery. Contrary to the pronounced declines in offshore areas, nearshore chlorophyll a concentrations increased or remained stable throughout time for most seasons. While no regional trends were observed in Lake Huron, nearshore trends were not homogeneous across regions in Lake Michigan. From 1998 to 2013, Chlorophyll a concentrations increased in nearshore areas in the southeast and northwest quadrants of Lake Michigan, most likely due the influence of large adjacent agricultural watersheds and warmer surface water temperatures that characterize these regions. These results were in contrast to the southwest region where nearshore chlorophyll a concentrations declined over the course of 16 years. Increases in nearshore chlorophyll a concentrations and subsequent increase in prey availability, may corroborate the recent reliance of nearshore resources by some fish species in the Great Lakes. The second aspect of this study focused on whether increased nearshore food web subsides would affect the condition of Bythotrephes along a nearshore to offshore gradient and regionally in Lake Michigan. No nearshore to offshore differences in lengths, RNA content, and relative fatty acid content were observed; however, regional differences were, again, prominent. Regional variation in surface water temperature facilitated larger sizes and overall condition of Bythotrephes on the eastern shores of Lake Michigan during the spring and summer. By the fall, surface water had warmed sufficiently lake-wide to allow for comparable size and condition in Bythotrephes across southern Lake Michigan. Overall, responses to oligotrophication at lower trophic levels have differed in both time and space in lakes Michigan and Huron. Further description of how lower trophic levels respond to oligotrophication can help improve our understanding of how nutrient abatement programs and the establishment of invasive filter feeders are influencing food webs across the Great Lakes.

Natural and synthetic estrogenic and androgenic compounds are continuously released into aquatic ecosystems. Exposure of teleost fishes to these contaminants can negatively impact sex differentiation and reproductive output. Specifically, development of gonadal intersex in gonochoristic (fixed sex) fish species has been studied extensively in relation to exposure to this class of compounds. The main objectives of this dissertation were to: 1) conduct field and laboratory studies to investigate the molecular signaling pathways behind the development of gonadal intersex; and 2) establish molecular biomarkers and assays for testing the ability of environmental pollutants to develop this condition using a battery of molecular, cellular and organ-level tools. First, we conducted a literature review to summarize all available articles reporting gonadal intersex in wild populations of gonochoristic fish (Chapter 1). We also included the limited information available on this topic in amphibians. We analyzed studies from across the globe, identified families and species with reported cases of gonadal intersex, and highlighted the contaminants often linked with this condition. In addition, we discussed the current knowledge of molecular signaling pathways behind the development of gonadal intersex and summarized molecular biomarkers tested and others that require further investigation. We then conducted a field study investigating the prevalence of testicular oocytes (TOs), the most prevalent form of gonadal intersex, in a sentinel freshwater fish species, the smallmouth bass Micropterus dolomieu, inhabiting the St. Joseph River and its tributaries in northern Indiana (Chapter 2). This constitutes the first study of this nature. Sites on this river were previously identified as having medium to high intersex induction potential based on contaminant quantification and estrogen equivalence estimations. We reported prevalence and severity of gonadal intersex reaching 100% in some sites, and significant decreases in prevalence and increases in severity of TOs occurrence after the spawning season. We evaluated changes in the transcription levels of several genes involved in sex differentiation and gonadal development. Significantly higher levels of vitellogenin ( vtg) transcripts were found in livers of males with TOs, but only when sampled in the spawning season. Further, we quantified contaminant levels in surface water to identify possible correlations between contamination levels and the observed prevalence of gonadal intersex. Multiple sites had detectable levels of endocrine disruptors, but no correlations with the prevalence or severity of TOs was recorded. In order to develop molecular biomarkers and assays that test contaminants’ ability to develop gonadal intersex, short-term laboratory exposures were performed using Japanese medaka Oryzias latipes, a fish model with a well-understood sex determination mechanism and high sensitivity to exogenous hormone exposure (Chapter 3). First, we identified a gene, ovary structure protein 1 (osp1), with strong female-specific expression during gonadal differentiation and observed a significant downregulation in its expression in females following short-term (10 d) exposure to a potent synthetic androgen, 17β-trenbolone. Importantly, this decrease in osp1expression was correlated with changes in ovarian phenotype, namely ovarian intersex, later in life. We decided to further utilize this promising molecular biomarker by incorporating it in a visual in vivo reporter assay for rapid detection of contaminants with estrogenic/androgenic potential (Chapter 4). For this purpose, we built a pOSP1-AcGFP (promoterOSP1-Aequorea coerulescens green florescence protein) Japanese medaka transgenic line with osp1 promoter region driving the expression of a reporter protein, AcGFP. After establishing this line, we tested its use in an in vivo visual reporter system for identifying estrogenic contaminants. Significant upregulation in fluorescence intensity was recorded in 30 d post hatch females following a 24 h exposure to 500 ng/L of a synthetic potent estrogen, ethinyl estradiol. Overall, our results support earlier findings suggesting that gonadal intersex is highly prevalent in impacted rivers across the US and that smallmouth bass are highly sensitive to developing this condition. We also conclude that hepatic and plasma VTG are promising biomarkers for diagnosing gonadal intersex, but only in males sampled during the spawning season. Our findings also support the hypothesis that molecular biomarkers, such as osp1 , are sensitive tools that can be used for early detection of the effects of contaminants with estrogenic and androgenic activity on fish and are ideal endpoints in wide-scale contaminant screening assays.

Grain entrapments and engulfments are one of most common hazards associated with grain storage facilities. Since the 1970’s over 1,880 incidents have been documented in agricultural confined spaces of which 65% of all recorded incidents were grain entrapments and engulfments. There have been several studies conducted on the contributing factors behind these incidents; however, there have been very few attempts to understand the environmental, physiological or psychological factors the victims experience while entrapped, engulfed, or extricated. This includes understanding how secondary injuries are caused by grain or during extrication by first responders. The research effort was divided into three segments. The first segment is a literature review to identify and better understand the environmental, physiological and psychological stresses that an individual might be exposed to during grain entrapment, engulfment or extrication. The second segment expands upon previous studies that involved vertical pull tests (Schwab, Ross, Piercy, McKenzie, & B.A, 1985; Roberts, Field, Maier, & Stroshine, 2015) by testing forceful extrication attempts under a wider set of variables, including different types of grains (corn, popcorn, wheat, oats, soybeans, canola seeds and sunflower seeds), depths of entrapment, pull angles (15°, 30°, 45°, 60°, and 75°), limb placement and grain moisture content (corn only). With the exception of the pull angle test, these experiments were conducted only in a small scale setting. Pull angle tests were conducted in a full scale setting using a full sized mannequin (185 lb) in corn and soybeans. This is an important study since grain bin roofs are not generally designed for 5,000 lb anchor points. In addition, the tensile force limits of a sheep spine were tested and compared to the force needed to extricate a mannequin. The third segment focused on measuring the actual pressure that a victim might experience by pushing wooden plates against grain (simulating a rib cage pushing against the grain) and measuring the force. These experiments also focused on localized forces on the spine and limbs and estimating forces generated when a test mannequin is extricated at different angles. The literature review provided a total of eleven factors that negatively impact a victim’s ability to survive a grain entrapment. The most important factor was asphyxiation (which includes aspiration, crush asphyxiation and postural asphyxiation). In 33 cases where the cause of death was medically reported, 63% cited asphyxiation. Another factor of notable importance is psychological, where it was found that stress could cause shortness of breath and chest pain and thus could be a contributing factor in death. In the extrication segment of the research, it was found that high moisture content could increase extrication forces by 39%. In addition, while shallow angles of pull did not significantly impact extrication force, pulling a victim at angles sharper than 45o degrees increased extrication forces by 22-44%. Lastly, the author found that the maximum tensile force that a spine can handle (1.65-2.48 kN) was in the same range of forces required to extricate a victim from between waist and shoulder depth. In the third segment of research, the author found that passive pressure on the victim was about four times larger than active pressure, thus a victim will experience four times more pressure in grain (while attempting to breath) than what a load cell measures. In conclusion, the best strategy to prevent or reduce the severity of injuries associated with grain entrapments remains prevention through compliance with accepted best workplace practices and current workplace safety regulations. It was determined that 94% of all grain entrapment and engulfment incidents were preventable. Regarding methods of victim extrication from grain entrapment it was concluded that there is a real and possible risk of causing secondary injuries, including spinal injury, if force is used to pull the victim from the grain. Reducing the pressure on the victim by removing the grain from around the victim is strongly recommended unless there are other significant medical issues that might reduce the likelihood of survival if extrication is not expedited.

The dopamine transporter (DAT) is a monoamine transporter that regulates dopamine (DA) neurotransmission by clearing DA from the synapse. DAT is acted on by a number of psychostimulant drugs, which block reuptake and potentiate DA signaling. Several inhibitors of DAT, both amphetamines and cathinones, also cause reversal of transport. Previous research has shown the importance of a conserved salt bridge in another monoamine transporter, the serotonin transporter, in amphetamine-induced efflux. In our studies here, we engineered a mutant Drosophila melanogaster DAT (dDAT D475N) designed to interrupt this salt bridge and examine the effects on amphetamine- and cathinone-induced efflux in vitro. Understanding the mechanism of action of structurally similar yet functionally diverse psychostimulants necessitates information about their binding sites at the transporter. A second mutant D. melanogaster DAT (dDAT D121G) was engineered to more closely mimic the substrate binding site of human DAT. By using already existing crystal structure data, compounds were computationally docked to the identified binding sites, allowing prediction of binding poses and binding free energies. dDAT D475N was found to have similar inhibition of uptake by synthetic cathinones relative to wild-type. No significant drug-induced efflux was observed in dDAT wild-type or dDAT D475N, while the D121G mutant restored efflux activity beyond that of human DAT. These results show that the presence of a salt bridge at the external gate of DAT, as well as a hydrophobic environment within the DA binding site, are important for drug-induced efflux.

Nanoparticles (NPs) are widely used in a myriad of commercial and industrial products making their entry to the environment a likely event. NPs have unique physical-chemical properties that result from their small size and high surface area to volume ratio, making them highly reactive and potentially toxic. In Chapter 1, we summarize the effects and mechanisms of metal-based NPs on the vascular system. In vitro studies have shown that NPs are anti-angiogenic because they cause inflammation, oxidative stress, and apoptosis of endothelial cells resulting in increased permeability and decreased proliferation and migration. Whole animal studies examining the effects of NPs on the vascular system are scarce and although the few available studies do not disproof the anti-angiogenic properties of NPs, the mechanisms and long-term effects of these changes are poorly understood. Considering the key physiological role of the vascular system, there is a need for more studies in this area. In Chapter 2, we show that blood proteins can quickly bind to the surface of NPs, which might greatly change their biological identity. We present novel data on the formation of a “protein corona” (PC) after incubation of polyvinylpyrrolidone-coated AgNPs (PVP-AgNPs, 50 nm) in fish (smallmouth bass) plasma. Both gender and length of incubation affected the types of proteins identified from PC. The most common proteins were related to immune function, red blood cell function and clotting, and lipid and cholesterol transport. Vitellogenin and zona pellucida egg proteins were only detected in PCs incubated with female plasma. We propose that fish plasma serves as a good media for the characterization of PC in future studies with NPs and that binding of egg proteins to the surface of PVP-AgNPs could result in enhanced movement of particles to developing oocytes and maternal transfer to developing embryos. In Chapter 3, vascular effects of PVP-AgNPs ( 60 nm) were evaluated on transgenic zebrafish (TG fli1a: EGFP) embryos. Exposure to 1 and 10 mg/L PVP-AgNPs during the period of vascular development caused a delay in vascular development; however, expression of genes within the vascular endothelia growth factor (VEGF) signaling pathway was enhanced. This apparent contradiction was explained by the induction of hypoxic conditions in the embryo via agglomeration of NPs on the surface of eggs. Hypoxia is a potent stimulant of the VEGF signaling pathway, but blood vessel development is not enhanced because no increased production in VEGF protein was observed, likely because of impaired translation due to AgNP toxicity to the endoplasmic reticulum. In Chapter 4, we evaluated the toxicity of a commercial product (socks) containing AgNPs. Importantly, we demonstrate that the toxicity of this leachate to zebrafish embryos was not due to AgNPs, but instead to the presence of unknown chemical(s). At the time this study was published, there was no data on the toxicity of AgNPs released from any commercial products. Overall, this set of studies advances our current understanding on the mechanisms of toxicity of AgNPs by providing novel molecular and whole animal data that can be used for future risk assessment for this emerging class of contaminants.

Bacterial and fungal resistance to conventional antimicrobials is a burgeoning global health epidemic that necessitates urgent action. Even more alarming, the development of new antimicrobials to treat these multidrug-resistant pathogens has not kept pace with the rapid emergence of resistance to current antimicrobials. Antimicrobial drug development through the traditional de novo process is a risky venture given the significant financial and time investment required by researchers and limited success rate of translating these compounds to the clinical setting. This has led researchers to mine existing libraries of clinical molecules in order to repurpose old drugs for new applications (as antimicrobials). The main aim of this research endeavor was to screen and validate approved drug libraries and small molecules for their antimicrobial activity against multidrug-resistant bacterial and fungal pathogens, includingStaphylococcus aureus and Candida albicans. The present study identified four approved drugs (auranofin, ebselen, simvastatin and celecoxib) that exhibited potent antimicrobial activity against multidrug-resistant bacterial and fungal pathogens. Notably, auranofin, an FDA-approved anti-rheumatic drug possessed excellent antibacterial activity against S. aureus and was found to exert its effect by inhibiting multiple biosynthetic pathways including DNA, protein and cell wall synthesis. Furthermore, auranofin was found to be efficacious in a mouse model of S. aureus systemic infection, as it significantly reduced the bacterial load in murine organs, including the spleen and liver. Ebselen, an organoselenium compound known to be clinically safe, exhibited potent anti-staphylococcal activity by inhibiting bacterial protein synthesis. Other approved drugs including simvastatin (anti-hyperlipedmic drug) and celecoxib (non-steroidal anti-inflammatory drug) also possessed anti-staphylococcal activity against various clinical isolates of S. aureus. Our study also revealed that three drugs (auranofin, ebselen and simvastatin) markedly reduced the production of major staphylococcal toxins including Panton-Valentine leucocidin (PVL) and α-hemolysin (Hla), thereby improving the treatment outcome against toxin-producing bacterial pathogens. Furthermore, all these drugs effectively reduced both the bacterial load and inflammatory cytokines in a mouse model of S. aureus skin infection. In addition to their antibacterial activity, auranofin and ebselen were found to possess potent antifungal activity against two major pathogens, Candida and Cryptococcus; they exerted their antifungal effect through inhibition of mitochondrial proteins (auranofin) and glutathione synthesis (ebselen) respectively. Additionally, these two drugs proved superior to control antifungals, as they reduced the fungal load in a Caenorhabditis elegans animal model. Taken altogether, the potent in vitro and in vivo antimicrobial activity (against bacterial and (or) fungal pathogens) of auranofin, ebselen, simvastatin and celecoxib indicates these four drugs have considerable promise to be successfully repurposed for use as antimicrobial agents.

Chapter 1 details a background of techniques used for modeling the blood-brain barrier (BBB). The BBB represents a diffusive barrier to both paracellular and transcellular movement of many compounds in and out of the brain. The main rate- limiting barriers of the BBB include exclusive tight junctions that prevent the movement of hydrophilic molecules through intercellular gaps, and efflux proteins in the membrane which pump many hydrophobic molecules back into the blood. In addition, the BBB contains metabolizing enzymes, including Cytochrome P450s. This barrier acts to protect the vulnerable tissues of the brain from harmful xenobiotics, but also can serve as a restrictive barrier for potential therapeutic compounds for the growing number of neurological diseases, including Alzheimer’s disease, Parkinson’s disease, stroke, depression, brain cancers, and many others. A number of in vitro cell screens have been established to mimic the BBB for permeation testing. Principally, the best model should include primary human brain microvessel endothelial cells (BMECs), however, due to the growing interest in BBB permeation and lack of tissues, the supply is limited. As an alternative method, many groups have investigated the use of primary animal BMECs, usually of murine, porcine, or bovine source. These models prove effective at restricting paracellular movement; however, one must question the effects of animal isoforms on modeling uptake, efflux, and metabolism of transcellular markers. This proves important as in vivo most, if not all, therapeutic compounds will cross the BBB transellularly. Consequently, much work has been done to establish immortalized human BMECs. These immortalized cell lines alleviate the high cost and lack of supply found with primary human cells, but potentially may serve as a better model for transcellular permeation over animal cell lines. Currently, the most widely characterized immortalized human BMEC cell line is the human cerebral microvessel endothelial cell line (hCMEC/D3). These cells express tight junction proteins, efflux proteins, cyp450 enzymes, and are conducive to in vitro testing. However, while these cells express tight junctions, their function is less than ideal and leads to a leaky monolayer which may allow faster permeation through the paracellular route or paracellular permeation of compounds that move transcellularly in vivo leading to poor prediction of BBB permeability. One method of investigating the reason behind these leaky tight junctions was to take a closer look at the BBB itself. Chapters 2 and 3 discuss the optimization and establishment of a direct contact coculture in vitro model in which both endothelial cells and astrocytes are plated on the apical side of the Transwell® allowing for more physiologically relevant signaling between the cells. Early results suggest decreased paracellular permeation in this configuration compared to endothelial monocultures and indirect cocultures. In addition, this setup should allow for easier transition to high-throughput equipment. Additional studies in Chapter 4 attempt to show the utility of a new mono-PEGylated Human Serum Albumin (HSA) as a potential enhancer of drug solubilization, permeation, and eventual cytotoxicty. Multi-gram batches of short (5 kDa) and long (20 kDa) PEG-HSA were synthesized with high efficiency (77%) and characterized in collaboration with Dr. Jonathan Mehtala and Dr. Alex Wei in the Department of Chemistry. Furthermore, effects of PEG-HSA on permeation of paclitaxel through peripheral and BBB in vitro cell models as well as changes in cytotoxicity against MCF-7 cells was investigated. Finally, Chapter 5 includes an investigation into the characterization, formulation, and in vitro/in vivo testing of a potent V-ATPase inhibitor known as Saliphenylhalamide. The V-ATPase is an endogenous protein that is responsible for acidifying intercellular compartments and has been targeted in the past with in vitro success for treatment of cancer and osteoporosis among other diseases. However, here we investigate its use as an anti-viral therapeutic as acidification of endosomes by the V-ATPase is thought to be a critical step in replication of alpha viruses. Initial characterization showed poor water solubility and acid liability. Therefore, two solubility enabling formulations were created and tested for in vitro permeability and in vivo murine pharmacokinetics. (Abstract shortened by ProQuest.)

Probiotics are live microorganisms which may confer health benefits on the host when administered in appropriate amounts. Numerous studies have shown that probiotics improve bone health in humans and rodents with less information available on the skeletal health of avians given probiotics. The objective of this study was to determine the effects of probiotic supplementation on bone health of egg-laying and meat-type chickens. Dietary supplementation of a multi-species based probiotic reduced the percentage of shell-less eggs beginning at 4 wk following treatment and increased tibial and femoral bone mineral density in egg-laying hens at 7 wk post-treatment. Similarly, bone mineralization accrual of the tibia and femur was improved in broilers subjected to daily cycling heating episodes fed the same probiotic for 6 wk. Broilers consuming the probiotic also had a lower incidence of lameness as characterized by an improved gait score and longer latency to lie. The effects of a single-species based probiotic fed to broilers beginning at 1 d of age on bone health were investigated. Bone mineral densities of the tibia and femur were increased at 43 d of age. Concomitantly, serum calcium concentrations were increased at 14 d of age, serotonin levels were up-regulated in the raphe nuclei, and the catecholamine concentrations of NE and DA were down-regulated in the hypothalamus at 43 d of age. With the exception of bone mineral density, the bone health of probiotic-fed broilers was maintained under elevated temperature as indicated by larger bones and higher bone mineral content of the tibia and femur at 43 d of age accompanied by reduced plasma concentrations of TNF-α, a pro-inflammatory cytokine. Dietary inclusion of probiotics is a useful strategy for improving skeletal health in chickens under both normal and elevated ambient temperature. Probiotics enhance intestinal absorption of nutrients like calcium and may reduce sympathetic activity, thus improving mineralization of bone. Under the condition of daily cycling heating episodes, probiotics increased bone growth and bone size of broilers perhaps via inhibition of bone resorption resulting from the down-regulation of TNF-α, thereby reducing systemic inflammation.

This research examined the lived experiences of small businesses in Northwest Indiana with the implementation of the Affordable Care Act. The interviews were conducted over a period of six months with an attempt to answer the question, “What was the impact of the Affordable Care Act on small businesses and industries in Northwest Indiana?” Additional research questions were, “Did the Affordable Care Act extend coverage to the uninsured, include other medical services that were not covered before, and reduce cost sharing and what lessons can be learned from the implementation of the Affordable Care Act for future policy implementation?” 15 participants from Northwest Indiana were interviewed separately for 30 to 45 minutes each. Based on the analysis of data from interviews and observations, seven major themes emerged. These included the old system needed to be fixed, the Affordable Care Act made things worse was a form of government interference, it increased premiums, resulted in fear, frustration, and confusion, had positive outcomes, and eliminated pre-existing conditions. The outcomes of this study confirmed that while the Affordable Care Act helped some people obtain health insurance by eliminate pre-existing conditions, there is a strong indication that it also resulted in some unwanted consequences. This contribution includes suggestions for future qualitative study on the impact of the Affordable Care Act on small businesses.

Terrestrial ecosystem plays a critical role in the global carbon cycle and climate system. Therefore, it is important to accurately quantify the carbon dynamics of terrestrial ecosystem under future climatic change condition. This dissertation evaluates the regional carbon dynamics by using upscaling approach, mechanistically-based biogeochemistry models and in situ and remotely sensed data. The upscaling studies based on FLUXNET network has provided us the spatial and temporal pattern of the carbon fluxes but it fails to consider the atmospheric CO2 effect given its important physiological role in carbon assimilation. In the second chapter, we consider the effect of atmospheric CO2 using an artificial neural network (ANN) approach to upscale the AmeriFlux tower of net ecosystem exchange (NEE) and the derived gross primary productivity (GPP) to the conterminous United States. We found that atmospheric CO 2effect on GPP/NEE exhibited a great spatial and seasonal variability. Further analysis suggested that air temperature played an important role in determining the atmospheric CO2 effects on carbon fluxes. In addition, the simulation that did not consider atmospheric CO2 failed to detect ecosystem responses to droughts in part of the US in 2006. The study suggested that the spatially and temporally varied atmospheric CO2 concentrations should be factored into carbon quantification when scaling eddy flux data to a region. The process-based ecosystem models are useful tools to predicting future change in the terrestrial ecosystem. However, they suffer the great uncertainty induced by model structure and parameters. The carbon isotope (13C) discrimination by terrestrial plants, involves the biophysical and biogeochemistry processes and exhibits seasonal and spatial variations, which may provide additional constraints on model parameters. In the third chapter, we found that using foliar 13C composition data, model parameters were constrained to a relatively narrow space and the site-level model simulations were slightly better than that without the foliar 13C constraint. The model extrapolations with three stomatal schemes all showed that the estimation uncertainties of regional carbon fluxes were reduced by about 40%. In addition, tree ring data have great potentials in addressing the forest response to climatic changes compared with mechanistic model simulations, eddy flux measurement and manipulative experiments. In the fourth chapter, we collected the tree ring isotopic carbon data at 12 boreal forest sites to develop a linear regression model, and the model was extrapolated to the whole boreal region to obtain the water use efficiency (WUE) and GPP spatial and temporal variation from 1948 to 2010. Our results demonstrated that most of boreal regions except parts of Alaska showed a significant increasing WUE trend during the study period and the increasing magnitude was much higher than estimations from other land surface models. Our predicted GPP by the WUE definition algorithm was comparable with site observation, while for the revised light use efficiency algorithm, GPP estimation was higher than site observation as well as land surface model estimates. In addition, the increasing GPP trends estimated by two algorithms were similar with land surface model simulations.

Well-developed replacement heifers provide a central foundation for the continued success of the dairy industry. Emphasis on improving pre-weaned calf nutrition has predominated in the industry, but opportunities exist to improve post-weaning heifer nutrition and management. We aimed to evaluate common feed management strategies seen in the industry and their effects on growth, feed efficiency (G:F), and rumen development of calves from birth to 8 mo of age using pen- and individually-fed animal trials. Little information exists regarding post-weaning performance and rumen development of calves fed conventional or high planes of nutrition pre-weaning; therefore, we evaluated two milk replacer feeding programs with two post-weaning diets differing in non-fiber carbohydrate (NFC) content. Overall, calves fed a high plane of nutrition pre-weaning with a low NFC post-weaning diet were 9.8 kg and 12.4 kg lighter than calves fed low or high planes of nutrition pre-weaning, respectively, with a high NFC post-weaning diet. Additionally, average daily gain (ADG) and frame height were increased for high NFC-fed animals regardless of pre-weaning treatment. Rumen development with respect to tissue morphology was similar between pre-weaning planes of nutrition in 12 wk old calves. However, 28 wk old animals previously fed a conventional milk replacer program had 20% greater papillae surface area compared to calves fed a higher plane of nutrition pre-weaning. Interestingly, rumen papillae morphology was similar between post-weaning diets and no interaction of pre- with post-weaning diet was observed. However, in a concurrent study evaluating low and high NFC diets using pen-fed heifers, feeding low NFC diets with added fat resulted in higher ADG and G:F compared to heifers fed high NFC diets despite similar dietary ME and CP content. Overall, these results suggest feeding diets with highly digestible carbohydrates to promote greater G:F and skeletal growth post-weaning, particularly when higher planes of nutrition are provided pre-weaning. As G:F improved when NFC and starch increased in the diet, we investigated increasing dietary concentrate proportions for growing heifers and the effects when switched to a high forage diet. Heifers fed 80% concentrate were the heaviest, tallest, and most feed efficient during the treatment period, but exhibited decreased in performance when switched to a high forage total mixed ration (TMR; 60% hay) compared to heifers previously fed 60% or 40% concentrate. Additionally, molar proportions of propionate and butyrate were greater when heifers were fed 80% or 60% concentrate, potentially influencing rumen development. Delivering of feed as a TMR is common practice on dairy operations, as nutrients are delivered consistently with increased labor efficiency. Additionally, ensiled forages are commonly included as the primary forage component in heifer diets, although growth and intake responses when feeding ensiled forages as compared to hay are limited and inconsistent. We compared feeding a common diet delivered by feeding hay and concentrate separately (HF), hay side-dressed with concentrate (SBS), and a TMR, observing that G:F of HF- and SBS-fed heifers was 8 to 10% greater overall compared to heifers fed a TMR. Additionally, HF-fed heifers were 13.5 kg heavier at the end of the study and had 5.6% greater DMI overall compared to SBS- and TMR-fed heifers. We also fed weaned heifers diets using mixed grass/legume forage preserved as hay or baleage as the only forage source to evaluate growth and efficiency. Heifers fed hay were 6.7 kg heavier than heifers fed baleage at the conclusion of the study. Heifers fed hay also consumed more DM and tended to have greater G:F than heifers fed baleage. As G:F was affected by forage preservation, feed delivery method, and carbohydrate inclusion in growing heifer diets, we postulated that rumen development may not be as mature as previously believed for heifers post-weaning. Greater understanding of the effects of feed management strategies on growing heifer performance has been achieved from our research, allowing more precise feeding recommendations and development of feeding programs to improve heifer development.

Post-traumatic Stress (PTS) symptomology includes ruminating thoughts and feelings around trauma, inability to feel and express emotions, and avoidance of things related to the traumatic event (American Psychiatric Association, 2000). Children and youth exposed to child maltreatment (abuse and neglect) are at higher risk of experiencing PTS. Extra-familial support, including peer support can reduce post-traumatic stress among youth (Pina et al., 2008). In the present study, Witnessing, Victimization, and Both Witnessing and Victimization due to Exposure to In-Home Violence and Peer Relationship Quality are evaluated as to their relative impact on PTS for children at risk for child maltreatment. Peer Relationship Quality is also tested as a moderator of the effects of violence exposures on PTS. Data come from the National Survey of Child and Adolescent Well-Being II (NSCAW II). Three waves of assessment were obtained starting in 2008 at 18 month intervals. A subsample of 2,151 children (2,071 children with non-missing values on the predictor, outcome and moderator variables) who were between the ages of 8 and 17 at any of the 3 waves was used. A three process latent linear growth model was estimated to assess PTS, Peer Relationship Quality and Exposure to In-Home Violence. Each of three Exposure to In- Home Violence constructs were assessed separately. Findings suggest that the average PTS at baseline was about 9.18 points (on a 32 point scale) and PTS declines by about 0.58 points every year on average. Additionally, baseline Peer Relationship Quality was predictive of baseline PTS with higher scores on Peer Relationship Quality associated with lower PTS at baseline. Moreover higher Peer Relationship Quality over time was associated with declines in PTS over time. The baseline effect of Exposure to In-Home Violence was positively associated with baseline PTS. So more Exposure to In-Home Violence was related to more post-traumatic stress. Change in witnessing violence at home over time and change in both witnessing and victimization over time were strongly positively associated with change in PTS over time. Peer Relationship Quality did not moderate the association between baseline Exposure to In-home Violence and PTS levels at baseline nor between baseline Exposure to In-Home Violence and change in PTS over time. These findings suggest several possible avenues for intervention for clinicians and help understand the dynamic associations between Exposure to In-Home Violence, Peer Relationship Quality and PTS in the population of children at risk for maltreatment.

Drawing from the stress process model and the cumulative disadvantage theory, this dissertation examined how childhood and later life stressors affected cognitive and subjective health in older adults. Using three main articles, this dissertation investigated (1) the effect of early parental loss on cognitive well-being in Chinese oldest old; (2) the effect of intergenerational socioeconomic mobility on cognitive and subjective health in advanced age; and (3) the effect of different dimensions of socioeconomic status and perceived financial strain on subjective health in later life. Data were derived from the 2002 and 2005 waves of the Chinese Longitudinal Healthy Longevity Survey. Ordinary least squares models and logistic regression models were used to estimate the stress-health association. Key findings of this dissertation include (1) losing a mother in early life predicted worse cognitive function in oldest old men; (2) experiencing downward intergenerational socioeconomic mobility was detrimental to cognitive and subjective health in advanced age, especially in oldest old women; and (3) perceived financial strain was associated with an elevated risk of reporting poor health for both urban and rural inhabitants. Findings of this dissertation suggest that traumatic events in early life and socioeconomic stressors in later life both exact a toll on Chinese older adults’ health, independent of their demographic characteristics and physical health conditions. This dissertation has policy implications for gender-specific healthcare.

The lack of literature examining sexual experiences of South Asian women in dating relationships has important implications for the healthy development of long lasting romantic relationships. It is important to understand South Asian women’s relationship experiences in the context of power and sexism (interpersonal power framework; Pulerwitz, Gortmaker, & DeJong, 2000) and the role of specific sociopolitical factors (e.g., discrimination). Understanding South Asian women’s experiences of power in a sociopolitical context will help professionals when working with them to develop healthier sexual relationships through therapy outreach, and community programming. I used a correlational, quantitative study to examine the associations between sexual relationship power, sexism, cultural values conflict, discrimination, and social support among a sample (N = 161) of South Asian women who are in current or recent sexually involved premarital relationships. I hypothesized that sexism, cultural values conflict, and discrimination (i.e., recent, lifetime, appraised) will contribute uniquely and negatively to sexual relationship power. I also hypothesized that social support will: a) contribute uniquely and positively to sexual relationship power and b) also moderate the relationship between the other independent variables (i.e., cultural values conflict, discrimination, and social support) and sexual relationship power. The results revealed that cultural values conflict, recent discrimination, and social support uniquely contributed to sexual relationship power in the hypothesized directions. Sexism, lifetime discrimination, and appraised discrimination did not uniquely contribute to sexual relationship power. Additionally, social support did not moderate the relationship between sexism, cultural values conflict, and discrimination with sexual relationship power. Implications for practice, limitations, and future research directions are discussed.

Twitter may be a data resource to support healthcare research. Literature is still limited related to the potential of Twitter data as it relates to healthcare. The purpose of this study was to contrast the processes by which a large collection of unstructured disease-related tweets could be converted into structured data to be further analyzed. This was done with the objective of gaining insights into the content and behavioral patterns associated with disease-specific communications on Twitter. Twelve months of Twitter data related to cancer, diabetes, and asthma were collected to form a baseline dataset containing over 34 million tweets. As Twitter data in its raw form would have been difficult to manage, three separate data reduction methods were contrasted to identify a method to generate analysis files, maximizing classification precision and data retention. Each of the disease files were then run through a CHAID (chi-square automatic interaction detector) analysis to demonstrate how user behavior insights vary by disease. Chi-square Automatic Interaction Detector (CHAID) was a technique created by Gordon V. Kass in 1980. CHAID is a tool used to discover the relationship between variables. This study followed the standard CRISP-DM data mining approach and demonstrates how the practice of mining Twitter data fits into this six-stage iterative framework. The study produced insights that provide a new lens into the potential Twitter data has as a valuable healthcare data source as well as the nuances involved in working with the data.

Medicare provides health care coverage for approximately 93% of non-institutionalized older adults. Compared to uninsured adults, Medicare beneficiaries have greater access to needed healthcare including preventative care. However, disparities in accessing needed health care still exist among Medicare beneficiaries. Prior research has described barriers to accessing needed health care among older Medicare beneficiaries, such as transportation and health system characteristics, but little is known about prevalence, risks, or health consequences of older Medicare beneficiaries delaying, forgoing, or having trouble accessing needed health care. The three studies included in this dissertation followed a nationally representative sample of older Medicare beneficiaries to describe the phenomenon of older adults delaying, forgoing, or having trouble getting needed care. Study 1 described the prevalence and risks of Medicare beneficiaries forgoing, delaying, or having trouble getting needed medical care. Estimates of the prevalence and risks for forgoing, delaying, or having trouble accessing needed care were estimated separately for five years beginning with 2006 and ending in 2010 using the Medicare Current Beneficiary Survey (MCBS). A meta-analysis was conducted to determine overall effect sizes for the five years of data. Study results revealed that about one in every nine older Medicare beneficiaries reported forgoing, delaying, or having trouble accessing needed health care. Beneficiaries most likely to report going without or having trouble getting needed care were more likely to be of minority status, younger, female, more educated, live in a non-metropolitan area, have a lower annual income, have no supplementary insurance, be in poorer health, and have multiple chronic conditions or disabilities. This study confirmed that disparities in accessing needed care exist among Medicare beneficiaries. The study findings also revealed that those most likely to delay, forgo, or have trouble accessing needed care are among the most vulnerable Medicare beneficiaries due to their multiple chronic conditions or disabilities. The second study examined whether reports of forgoing, delaying, or having trouble accessing needed health care was prognostic of receiving an influenza vaccination in the following year. This study followed the 2006-cohort and 2007-cohort from the 2007 Medicare Current Beneficiary Survey to determine if receipt of the influenza vaccination in 2008 was associated with forgoing, delaying, or having trouble accessing needed health care. Nearly one in every four older Medicare beneficiaries reported not receiving an influenza vaccination. Those who reported forgoing, delaying, or having trouble accessing needed health care were significantly less likely (OR = 0.749; 95% CI = 0.609, 0.922) to receive an influenza vaccination the following year. Findings suggest that forgoing, delaying, or having trouble accessing needed healthcare increases risk for vaccination non-compliance; which in turn can increase risk for experiencing adverse health outcomes associated with influenza. The results of this study suggest that more proactive measures may be needed to increase vaccination rates among older adults who forgo, delay, or have trouble accessing needed health care. Study 3 examined whether reports of forgoing, delaying, or having trouble accessing needed care were prognostic of hospital admissions among older Medicare beneficiaries. Number and length of hospital admissions in 2010 were determined for respondents to the 2009 MCBS survey. Results revealed that forgoing, delaying, or having trouble accessing needed care was not prognostic (Hazards Ratio = 0.905; 95% CI = 0.722, 1.134) of future hospital admissions after adjusting for other risk factors associated with hospitalizations. Findings suggest that there may be potential confounding between forgoing, delaying, or having trouble accessing needed health care and hospital admissions. Further work should be considered to examine potential confounders and/or other health outcomes. The three studies in this dissertation improve our understanding of the prevalence, risks, and consequences of older Medicare beneficiaries forgoing, delaying, or having trouble accessing needed health care. The findings will inform the importance of developing interventions or policies aimed at improving older Medicare beneficiaries’ access to needed health care.

Many public health organizations have proposed and implemented campaigns and policies involving graphic ads or warnings to deter individuals from smoking. The current study evaluated how high-cigarette dependent smokers evaluate these messages compared to low-cigarette dependent smokers using a mixed, 3 x 2 factorial design. A pilot study involving focus groups, interviews, and evaluation questionnaires (N=10) was conducted to classify graphic threat appeals and develop stimulus materials for the primary study. For the primary study, three graphic threat appeal conditions were evaluated by Amazon Mechanical Turk (MTurk) users (N=200). Findings suggested that the graphicness of the images significantly influenced arousal, aversive response, and persuasion. However, high-cigarette dependent smokers evaluated messages as less aversive than low-cigarette dependent smokers to the graphicness in messages. Additionally, low-cigarette dependent smokers may be at greater risk for experiencing cognitive overload and reallocating cognitive resources to defensive responses. Implications of these findings and recommendations for future research and message design are discussed.

Purpose: The long-term goal of this research is to determine the feasibility of using near infra-red light to stimulate drug release in metastatic lesions within the brain. In this work, we focused on developing the tools needed to quantify and verify photon fluence distribution in biological tissue. To accomplish this task, an optical dosimetry probe and Monte Carlo based simulation code were fabricated, calibrated and developed to predict light transport in heterogeneous tissue phantoms of the skull and brain. Empirical model (EM) of photon transport using CT images as input were devised to provide real-time calculations capable of being translated to preclinical and clinical applications. Methods and Materials: A GPU based 3D Monte Carlo code was customized to simulate the photon transport within head phantoms consisting of skull bone, white and gray matter with differing laser beam properties, including flat, Gaussian, and super-Gaussian profiles that are converging, parallel, or diverging. From these simulations, the local photon fluence and tissue dosimetric distribution was simulated and validated through the implementation of a novel titanium-based optical dosimetry probe with an isotropic acceptance and 1.5mm diameter. Empirical models (EM) of photon transport were devised and calibrated to MC simulated data to provide 3D fluence and optical dosimetric maps in real-time developed around on a voxel-based convolution technique. Optical transmission studies were performed using human skull bone samples to determine the optical transmission characteristics of heterogeneous bone structures and the effectiveness of the Monte Carlo in simulating this heterogeneity. These tools provide the capability to develop and optimize treatment plans for optimal release of pharmaceuticals to metastatic breast cancer in the brain. Results: At the time of these experiments, the voxel-based CUDA MC code implemented and further developed in this study had not been validated by measurement. A novel optical dosimetry probe was fabricated and calibrated to measure the absolute photon fluence (mW/mm2) in phantoms resembling white matter, gray matter and skull bone and compared to 3D Monte Carlo simulated data. The TiO2-based dosimetry probe was shown to have superior linearity and isotropicity of response to previous Nylon based probes, and was better suited to validate the Monte Carlo using localized 3D measurement (< 25% systematic error for white matter, gray matter and skull bone phantoms along illumination beam axis up to a depth of 2cm in homogeneous tissue and 3.8cm in heterogeneous head phantom). Next, the transport parameters of the empirical algorithm was calibrated using the 3D Monte Carlo and EMs and validated by optical dosimetry probe measurements (with error of 10.1% for White Matter, 45.1% for Gray Matter and 22.1% for Skull Bone phantoms) along illumination beam axis. Conclusions: The design and validation of the Monte Carlo, the optical dosimetry probe and the Empirical algorithm increases the clinical feasibility of optical therapeutic planning to narrow down the complex possibilities of illumination conditions, further compounded by the heterogeneous structure of the brain, such as varying skull thicknesses and densities. Our ultimate goal is to design a fast Monte Carlo based optical therapeutic protocol to treat brain metastasis. The voxelated nature of the MC and EM provides the necessary 3D photon distribution to within 25% error to guide future clinical studies involving optically triggered drug release.

Dietary fat provides essential nutrients, contributes to energy balance, and regulates blood lipid concentrations. These functions are important to health, but can also become dysregulated and contribute to diseases such as obesity, diabetes, and cardiovascular disease. The small intestine absorbs dietary fat through an efficient multi step process of digestion, uptake, metabolism, and secretion or storage. When dietary fat is taken up by the absorptive cells of the small intestine, enterocytes, it can be secreted into circulation where it contributes to blood lipid levels or temporarily stored in cytoplasmic lipid droplets (CLDs). The objective of this dissertation is to investigate the presence and metabolism of CLDs in the intestine. Dietary fat absorption by the small intestine is a multi-step process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption. We also investigated the effect of diet induced obesity on CLD morphology and associated proteins. It is currently unclear what effect diet induced obesity (DIO) has on the balance between dietary fat storage and secretion. Specifically, there is limited knowledge of how DIO affects the level and diversity of proteins that associate with CLDs and regulate CLD dynamics. In the current study, we characterize CLDs from lean and DIO mice through histological and proteomic analyses. We demonstrate that DIO mice have larger intestinal CLDs compared to lean mice in response to dietary fat. Additionally we identified 375 proteins associated with CLDs in lean and DIO mice. We identify a subgroup of lipid related proteins which are either increased or unique to the DIO CLD proteome. These proteins are involved in steroid synthesis pathways, TAG synthesis, and lipolysis. This analysis expands upon our knowledge of the effect of DIO obesity on the process of dietary fat absorption in the small intestine. Lastly, we investigated the role of one of the newly identified CLD associated proteins on dietary fat absorption. Little is known of the role of Acsl5 in the metabolism of dietary fat in enterocytes. To understand the role of Acsl5 in dietary fat absorption, we challenged WT and Acsl5-deficient mice with acute and chronic dietary fat challenges. Despite no evidence of quantitative fat malabsorption in the absence of Acls5, we found significantly reduced TAG storage and secretion by enterocytes compared to WT mice. Interestingly, Acsl5-deficient consumed more food, but did not gain more weight compared to WT mice during two weeks of HFD feeding. Furthermore, we found that Acsl5 localizes to the nucleus after an acute dietary fat challenge. Together this data suggests that although Acsl5 is not essential for quantitative dietary fat absorption, it plays a role in intestinal metabolism of dietary fat. The results presented in this dissertation provide new knowledge of regulators of CLD metabolism. The long term objectives of these studies are to identify mechanistic details of the process of intestinal dietary fat absorption. Understanding the process of dietary fat absorption will provide new treatments for the prevention of diseases associated with dyslipidemia.

There is ample precedent for the use of 3D animation in education, though little research on specific applications in the field of medicine. An experiment was carried out to assess the suitability of 3D animation for educational purposes in emergency medicine courses. Two groups of experimental participants were assessed on their ability to respond to three emergency medical situations in simulated test scenarios. Both groups received equivalent information on how to treat the three different medical emergencies. The control group received the information in traditional lecture format. The experimental group instead received the information from a set of 3D animations. Participants were assessed according to the speed and accuracy with which they administered proper treatment as well as their ability to complete every step of treatment and to execute these steps in the correct order. The 3D animation and lecture groups were not generally found to score differently from a statistical perspective. Possible causes are provided.

Standard curves for a Perkin Elmer TriCarb 2800 liquid scintillation detector (LSC) and a Ludlum 3030p Passivated Implanted Planar Silicon detector have been developed and utilized for studying the dose distribution of depleted uranium (DU) within zebrafish. The DU source was crystallized uranyl nitrate (N2O8U•6H2O) solution, normally used for staining in electron microscopy with a manufactured average specific activity of 0.3 uCi/g. Zebrafish, both larvae and adults, were exposed to three different mass concentrations, dissected, dissolved and counted using an LSC. The counts were compared to the standard curve correlating the measured activity to that of the mass absorbed. It was found that the larvae were more tolerant to the toxicity of the DU by almost a factor of 10 showing survival up to 200 ppm where the adults had zero survival when exposed to concentrations above 20 ppm. The absorbed DU was observed to concentrate more heavily in the skeletal structure and the blood containing organs (liver and heart) when comparing the relative mass concentrations observed in each organ compared to that of the whole fish exposed to the same concentration. The highest absorbed dose rate was found in the skeletal system at 3.5 mGy/d followed by the blood containing organs at 2.2 mGy/d when exposed to 20 ppm DU. It was also noted that the bioconcentration factors (BCF) of the adult zebrafish followed the same trend observed in similar studies. As the mass concentration of DU was lowered, the BCF calculated for fish exposed increased with a BCF of 130.6 found for those exposed to 20 ppm U and a BCF of 774.2 for fish exposed to 2 ppm. This method shows to present a suitable way of developing a dose distribution for DU along with similar isotopes which will be instrumental in studying the long term effects of more specific exposures to natural radioactive metals combined with other common environmental exposures.

Both epidemiological and clinical evidence support the notion that polyphenol rich foods and beverages may modify glycemic response, glucose homeostasis and subsequent risk of Type-2 diabetes. In vitro evidence typically derived from experiments with pure phenolics and phenolic rich extracts have pointed to this benefit being associated with two potential mechanisms: (1) the ability of specific polyphenolics to inhibit carbohydrate digestion (amylase and glucosidase) and (2) polyphenolic inhibition of intestinal glucose transport. While the high potential of these activities is evident, little is actually known regarding the extent to which these benefits are extendable to the actual food matrix these phenolics are naturally present in. Further, the extent to which co-consumption of polyphenol rich foods may actually result in decreased glycemic response from a mixed meal remains mostly unknown. Considering these limitations, additional insights are required in order to advance knowledge on the benefits of polyphenolics on glucomodulatory mechanisms and translation of these insights into meaningful recommendations and products for consumers. With this in mind, the objectives of these studies were to determine the extent to which phenolic-rich foods (grapes and potatoes) exert glucomodulatory properties in model food systems using in vitro and in vivo assessments. First, mechanisms associated with polyphenol rich extracts or model foods on carbohydrate intestinal digestion and glucose transport were investigated in vitro using a three-stage in vitrodigestion model coupled to the Caco-2 human intestinal cell model. Components of this model used individually or in combination allowed for assessment of the two main mechanistic steps in phenolic modulation of glycemic response (starch digestion and glucose transport) in the context of interactions with bioaccessible phenolics. Additionally, the ability of the coupled in vitro digestion/Caco-2 model to predict in vivo outcomes was assessed. The first study compared the ability of 100% Niagara or Concord grape juice (GJ) phenolics to modify carbohydrase activity and intestinal glucose transport relative to a sugar sweetened beverage. While grape juices remain a major dietary source of phenolics, they are also well recognized to be naturally high in sugar content. Insights into the ability of natural fruit phenolics to modify glycemic response of grape juice were investigated in vitro. Also, in consideration that 100% GJ is consumed with meals, the extent to which modulation of carbohydrate digestion and intestinal absorption by GJ phenolics can be extended to a carbohydrate rich meal was evaluated. In the first experiment, inhibition of α-amylase and α-glucosidase by GJ extracts (300 and 500 μM total phenolics) and ability of GJ extracts (10 to 100 μM total phenolics) to modulate labelled glucose and fructose transport across Caco-2 intestinal cell monolayers compared to a phenolic-free control were determined. GJ extracts decreased α-glucosidase, but not α-amylase activity at both concentrations tested. Further, glucose and fructose transport were significantly (pin vitro with a starch-rich model meal. Resulting aqueous digesta (AQ) from both experiments were used to assess impact of bioaccessible GJ phenolics on carbohydrate digestion and glucose transport. Concord and Niagara GJs significantly decreased in vitro gastrointestinal digestion of carbohydrate from model meal compared with a sugar-matched control. Further, d7-glucose transport from AQ fraction of GJ and co-digested GJ and carbohydrate-rich meal across Caco-2 human intestinal cell monolayers was significantly decreased compared to phenolic-free sugar-sweetened control. The second study evaluated potential for phenolics from starch rich white, purple, or red potatoes to modulate carbohydrate digestion or glucose transport in a Caco-2 intestinal cell model. Potato phenolic extracts (300 μM) had no impact on α-amylase activity, and marginally decreased α-glucosidase activity. However, potato phenolic extracts (25-100μM) did decrease d7-glucose transport compared to phenolic-free control. Interestingly, whole potato phenolic extracts reduced glucose transport to a greater extent compared to those from potato peel. To determine if results from aforementioned in vitro assays are predictive of effects in vivo, a pilot clinical study (n=11) was completed to assess differences in acute blood glucose response and gastric emptying following consumption of phenolic-rich purple and red potato chips compared to white potato chips (50g available carbohydrate) containing lower level of total phenolics. Blood glucose levels were measured for up to two hours. Peak blood glucose levels were lower for pigmented chips, especially purple chips, compared to white chips without any significant changes in gastric emptying. These results suggest that potato phenolics may play a role in modulation of intestinal glucose transport and that these effects are translatable to consumer products such as potato chips. Taken together, these data support the notion that phenolics intrinsic to select foods have the ability to modify glycemic response through alteration of glucose transport and to a certain extent starch digestion. Therefore, it is likely that observed benefits associated with consumption of phenolic-rich foods and 100% fruit juices, as a part of an overall healthy diet, may be associated with the ability of intrinsic and bioaccessible phenolics to modify glycemic response. Future research that focuses on hypoglycemic effects of phenolic-rich foods should be larger scale and should evaluate a greater variety of phenolic-rich foods in order to better understand the extent to which phenolic class and food matrix impact hypoglycemic effects. Regarding meal-effects, a pilot clinical study should be completed to validate in vitro results and to provide information as to what degree various types of meal patterns alter glycemic effects of phenolic-rich foods. Such information can be leveraged in the development of phenolic-rich food products that have post-prandial glycemic effects and for making recommendations of dietary choices which may result in improved glucose homeostasis.

Flawed U.S. federal regulation of chemicals has resulted in a materials market that undervalues human and environmental health in favor of the more traditional attributes of price, performance and aesthetics. In the building products industry, global, dynamic supply chains and proprietary information concerns further complicate the task of assessing the material health of products. Voluntary material health programs in the green building industry are intended to incentivize the manufacture and selection of safer products by getting companies to gather and assess ingredient, hazard and risk information from their supply chain. Building product manufacturers considered early adopters of the main material health programs of interest were interviewed and surveyed in order to identify the barriers they face to further program adoption and disclosure of product content and hazard information. The research reinforced findings that data collection requirements should be further aligned between different material health programs in order to streamline the process for manufacturers. Release of appropriate levels of information for consumers is also crucial to incentivizing informed decision-making. Supplier engagement and consumer education were identified as pathways to accelerating the demand and release of better information. Addressing these barriers is important to progress, as voluntary measures are likely to remain the most efficient pathway to a healthier materials market.

Tablets are the most popular solid dosage form in the pharmaceutical industry because they are cheap to manufacture, chemically and mechanically stable and easy to transport and fairly easy to control dosage. Pharmaceutical tableting operations have been around for decades however the process is still not well understood. One of the common problems faced during the production of pharmaceutical tablets by powder compaction is sticking of powder to the punch face, This is known as 'sticking'. A more specialized case of sticking is picking when the powder is pulled away form the compact in the vicinity of debossed features. In the pharmaceutical industry, picking is solved by trial and error which is an expensive, labor intensive and time consuming affair. The objective of this work was to develop, validate, and implement a modeling framework for predicting picking in powder compacts. The model was developed in Abaqus a commercially available finite element package. The resulting model was used to investigate the influence of debossed feature geometry viz. the stroke angle and degree of pre-pick, and, influence of lubricant on picking. (Abstract shortened by ProQuest.)

The aim of the current study was to investigate the relationships between phonological awareness (PA) skills, types of speech sound errors, and nonword repetition skills. Ten preschoolers with typically developing speech (TD) and ten preschoolers with speech sound disorder (SSD), aged 4;0 (years; months) to 6;6 participated in the study. Eligible participants did not present with neurological, cognitive, or developmental disabilities such as cleft palate or autism spectrum disorder. We calculated the correlation between PA skills and nonword repetition performance of the children. In addition, a regression model was used to evaluate the degree to which phonological awareness skills could be predicted by the types of speech errors produced by the participants (typical speech errors, atypical speech errors, and distortions). Nonword repetition was significantly correlated with performance on the PA test, such that in general, participants who obtained poorer nonword repetition scores were found to have poorer PA skills. With regards to error types and PA skills, atypical errors predicted 12.5% of the variance in PA skills among TD participants. However, in children with SSD atypical errors did not contribute significant and unique variance to PA skills after controlling for age and nonverbal IQ. This data suggests that PA skills cannot be only inferred through the use of other measurements such as the SRT or speech sound errors produced.

Lead (Pb) is a toxicant well known for its effects on almost every organ system in the body. Pb use in industry has declined since removal of Pb from gasoline, but many developing countries still have significant use of Pb. Exposure to Pb has been linked with diseases causing neurodegeneration and thus have lasting effects long after the initial exposure. Another metal, strontium (Sr), has been linked with bone disease in particular situations and shown to have uses in treating osteoporosis as a supplement. However, there are no studies of the effects of Sr using a meaningful biomarker. The most commonly used biomarkers for Pb and Sr exposures are blood Pb and Sr; however, blood tests are unable to identify long-term exposure levels due to the short half-life of these metals in blood. Bone stores of Pb and Sr have a half-life of years to decades and serve as a biomarker of long-term exposure. X-ray fluorescence has been used to measure bone Pb and Sr. However, current systems have limitations with radioisotope sources, bulky equipment, and long measurement times. A portable XRF device capable of measurement of bone Pb and Sr, overcomes the limitation of the current systems and has been developed in this work. The detection limit of the portable XRF for bone Pb and Sr was found to be 11 ppm and 5 ppm respectively at 5 mm of skin thickness. The portable XRF will have limitations of measurement based on an individual’s skin thickness. The device was calibrated using standard phantoms and validated with in-lab samples, which demonstrated good agreement between KXRF and portable XRF measurements with strong correlations between goat bone, cadaver bone, and phantom measurements. In a population study of Pb poisoned children the portable XRF was further validated and a significant correlation between KXRF measured bone Pb and portable XRF measured bone Pb was identified; however, the device had limitations based on anatomical differences unaccounted for in children from our calibration. Adaptations of our calibration to account for the differences in children’s bone can be used to further improve on the results we obtained. Pb biokinetics was studied in these children, and the blood Pb half-life in the children was calculated to be about 10 days, which is much short than the 30 day half-life identified for adults. Bone Sr was measured in these children and a significant correlation with age was identified, indicating the Sr accumulates in bone. A novel high-energy x-ray tube based KXRF measurement system was tested for its feasibility of in vivo measurement of metals in bone using Monte Carlo (MC) simulation. The novel system shows a combination of the advantages of the portable XRF with a smaller scale device, x-ray tube source, and room temperature detector, as well as the advantages of the KXRF of minimal soft tissue signal degradation with more applicability to a wider range of populations. This device, with an optimized x-ray tube and uranium target of 0.056 mm, was found to have a detection limit for bone Pb measurement of about 3.6 ppm and could be adapted for measurements of multiple metals

Background: In the United Sates (US), more than 400,000 people have end stage renal disease (ESRD) or chronic kidney disease (CKD) stage 5D requiring in-center hemodialysis (HD), and approximately $35 billion is spent annually in healthcare costs. Maintenance HD patients with ESRD have increased protein (1.2 g/kg/d) and energy (30-35 kcal/kg/d) requirements, but the average protein and energy intake in these patients is only 0.8-1.0 g/kg/d and 20-25 kcal/kg/d, respectively. Subsequently, protein-energy wasting (PEW) is common, affecting up to an estimated 75% of maintenance HD patients, and low protein and energy intake are one of the main contributors of PEW. Consequences of PEW range from diminished quality of life (QoL) to increased mortality rate, and high protein meals during HD may help prevent PEW. However, eating during HD is often discouraged in US dialysis centers due to possible adverse events, particularly postprandial hypotension. The aim of this pilot study was to determine the effects of high protein meals during HD on symptomatic intradialytic hypotension (SH). Methods: 19 HD patients were recruited from 2 shifts (∼10AM-2PM, MWF & TTS) at one dialysis center for a 9-week, non-randomized, parallel arm study. Patients in the intervention group (N = 10) received meals with 30 grams of protein and one-third of sodium, potassium, phosphorus, and fluid recommendations according to Kidney Disease Outcomes Quality Initiative (KDOQI) for 25 dialysis sessions. Patients in the control group (N = 9) received a control social interaction. Blood pressure data from 2 months prior to the initiation of study and during the study (25 sessions each) were collected. Secondary outcomes included serum/plasma biochemistries (monthly renal laboratories to assess nutritional status and electrolyte balance), dialysis compliance, fluid retention, sleep, and other QoL measures. The difference in the frequency of SH events within groups for 2-month pre-study and during study, was determined by Wilcoxon signed-rank test, and between group differences were determined by Wilcoxon rank-sum test. Other outcomes were assessed using repeated measures ANOVA with fixed effects for group, time, and group × time interaction, and random effect for subject nested within group. Results: One patient in the intervention arm withdrew from the study upon discontinuation of dialysis. Thus, N = 9 patients per group completed the study and are included in the analyses. Patients were 62 ± 16 years old, 55% female, and had been on dialysis for 3.4 ± 2.6 years. In the intervention group, there were 4 SH events in 3 patients over 25 dialysis sessions in the pre-study period and 12 SH events in 4 patients over 25 dialysis sessions during the study period. In the control group, there was 1 SH event in 1 patient pre-study and 5 SH in 4 patients during study. Change in the frequency of SH events from pre-study to during study was not different between groups (P = NS). The average lowest mean arterial pressure (MAP) was significantly higher in the control group compared with the intervention group, but difference between pre-study and during study was not significant for either group. Serum albumin and pre-albumin did not change throughout the study and when compared with values obtained during the pre-study period in either group. There were no significant improvements in QoL with the meal intervention, but results showed that HD patients had poorer quality of life and sleep quality compared with the general US population. When patients were asked “how interested would you be in receiving nutritious meals during dialysis?” at the end of study, 70% of patients responded ≥ 4 (5-point scale, 5 = very interested; P = NS between groups). Conclusions: These pilot data suggest that meals during HD do not increase the frequency of SH events. Additionally, patients generally have positive attitudes towards receiving meals. However, changes in nutritional status indicators, electrolyte balance, and QoL were not observed in either group. Larger, longer-term studies are needed to confirm these results along with effects on nutritional and clinical outcomes in HD patients.

Vocal loading adversely affects the healthy larynx. The negative effects of vocal loading are thought to be exacerbated in dry environments, noisy environments, using non-habitual speaking patterns, and voice quality. Advancing age is also thought to be a risk factor for the negative effects of loading. To systematically tease out the effects of these factors on the healthy larynx, three different experiments were conducted. In each experiment, healthy participants produced 45-minutes of child-directed speech. In experiment 1, older, healthy adults produced loud child-directed speech, in the presence of background noise, in both low and moderate humidities, and voice was assessed. In experiment 2, young, healthy adults produced loud child-directed speech, in the presence of background noise, in both low and moderate humidities, and voice was assessed. In experiment 3, young, healthy adults produced child-directed speech using low-effort whisper quality and voice was assessed. In each experiment, voice measures included Phonation Threshold Pressure, Cepstral Peak Prominence, self-perceived phonatory effort, and self-perceived vocal tiredness. These voice measures were collected at set points of the frequency range. Our data suggest that the aging larynx is negatively affected by 45-minutes of loud child-directed speech and that humidification is beneficial in reducing these negative effects. Younger adults are also negatively affected by 45- minutes of loud child-directed speech, but not whispered speech. Increasing ambient humidity does not minimize these effects. The adverse effects of loud speech are much greater than whispered speech. Overall, these data increase our understanding of factors that load the larynx and lay the foundation for developing clinical tests to identify speakers who are susceptible to voice problems.