Previous in vitro studies have shown that shark cartilage extracts stimulate human leukocytes to release significant levels of TNFα, a cytokine typical of a Th1 immune response. The purpose of this study was to investigate further the effects of shark cartilage on cellular immune function, particularly cell proliferation, apoptosis, and IL-4 and INFγ production. The viability and proliferation of cell cultures grown in the presence of shark cartilage extract was not significantly different from unstimulated control cultures or those stimulated with mitogens (Con A, PMA, LPS), respectively. The effect of shark cartilage on apoptosis was determined by microscopic analysis of morphological apoptotic changes and by the detection of DNA fragmentation observed as characteristic ladder formation in agarose gel electrophoresis. While DNA fragmentation could not be demonstrated for cartilage-stimulated cells, characteristic morphological changes, indicative of apoptosis, were observed in leukocytes following incubation with shark cartilage extract. A statistically significant difference was not noted in the number of apoptotic cells present in cartilage-stimulated leukocytes and those stimulated with 0.5 μM/ml of staurosporine, suggesting that apoptosis is induced in the presence of cartilage extract. Culture supernatants of cartilage-stimulated leukocytes were assayed for IL-4 and IFN-γ by enzyme-linked immunosorbent assay (ELISA). Although a low level of IL-4 and INFγ was detected in culture supernatants of Con A and PMA stimulated cells it was not significantly different from that of unstimulated control cultures. Thus the significance of the absence of detectable IL-4 or INFγ in supernatants of cartilage- stimulated cultures could not be determined. However, as previously shown, supernatants did contain TNFα. Results of the study did not show a definitive pattern of cytokine production, characteristic of either a Th1 or Th2 type immune response.

Critical values are laboratory values that represent a life-threatening condition for which there is a treatment available. Laboratories make immediate notifications to ordering providers when critical values are identified so that they may quickly act to initiate a treatment for their patient. The majority of laboratories apply the inpatient critical value list to the outpatient setting, although there are many differences between an acutely ill inpatient population and an ambulatory outpatient population. The goal of this study was to determine if providers responded to the critical values in the outpatient setting and to determine if there was a difference in outcome indicators when providers responded to notifications and when they did not respond to notifications. Data for 673 critical value notifications for PT/INR, Digoxin, and Glucose results were collected from Riverside Health System’s five laboratories. Analysis suggested that the inpatient critical value lists and thresholds may not be appropriate to apply to the outpatient setting. In this study of 637 critical value notifications, providers chose not to respond to 25.7% of critical value notifications. Providers were more likely to respond to PT/INR and Digoxin critical value notifications that glucose critical value notifications. None of the cases for either of the three tests that went without a provider response resulted in death or serious harm to a patient, indicating that the critical value thresholds do not meet the definition of a critical value in the outpatient setting. In the future, laboratories should explore the utilization of a different critical value list and thresholds for the outpatient setting based upon patient outcomes.

Cardiovascular disease is now the leading cause of death worldwide. Serum amyloid A (SAA), a positive acute phase reactant, along with C-reactive protein is used clinically as a marker of cardiovascular disease risk. However, recent data has shed light on a possible causal role of SAA in the development of atherosclerosis, the most pervasive form of cardiovascular disease. Several inflammatory diseases such as diabetes and obesity are known to confer increased risk of developing cardiovascular disease. Individuals with these diseases all have modest but persistent elevation of SAA. To determine if SAA caused the development of atherosclerosis, apoe-/-chow fed mice were injected with either an adenoviral vector expressing human SAA1 (ad-hSAA1), a null adenoviral vector (ad-Null) or saline. Human SAA levels rapidly increased, albeit briefly then returned to baseline within 14 days in mice that received ad-hSAA1. After 16 weeks, mice that received ad-hSAA1 had significantly increased atherosclerosis compared to controls on the aortic intimal surface (p<0.0001), aortic sinus (p<0.05) and the brachiocephalic artery (p<0.05). According to the “response to retention” hypothesis; lipoprotein retention by vascular wall proteoglycans is a key initiating event in the development of atherosclerosis. We previously reported that SAA-stimulated vascular smooth muscle cells expressed biglycan with increased glycosaminoglycan chain length and increased binding affinity for low density lipoprotein. To further test the role of biglycan on the development of atherosclerosis we generated biglycan transgenic mice. These mice were crossed to the ldlr-/- mouse on a C57BL/6 background and fed a pro-atherogenic western diet for 12 weeks. There was a significant increase in atherosclerotic lesion area on the aortic intimal surface (p<0.05) and the aortic sinus (p<0.006), as well as a significant correlation between vascular biglycan content and aortic sinus atherosclerotic lesion area (p<0.0001). These data demonstrate that transiently increased SAA resulted in increased atherosclerosis compared to control mice, possibly via increased vascular biglycan content. In support of this we found that biglycan transgenic mice had significantly increased atherosclerosis compared to wildtype controls, likely through increased lipid retention in the vascular wall.

Complement is activated via two pathways: classical (CCP) and alternative (ACP). The CCP has been demonstrated in the nurse shark. The ACP has not been demonstrated in any cartilagenous fish. Nurse shark serum was evaluated for complement activity by its ability to lyse heterologous erythrocytes. As CCP activity requires calcium and magnesium, activity of shark serum chelated with EGTA (a selective calcium chelator) or EDTA (a chelator of calcium and magnesium) was assessed. Activity remained in serum chelated with EGTA but not EDTA. Furthermore, activity of chelated serum was enhanced by added magnesium. Activation of shark complement by activators of mammalian ACP (zymosan, LPS, inulin, CVF) was assessed. Complement was activated by zymosan and LPS. Immunoblots were employed, with limited success, to demonstrate complement proteins in nurse shark serum. This study unequivocally demonstrates that the ACP is present in the primitive nurse shark.

Neurogenesis is an important and vastly under-explored area in reptiles. While the ability to generate new brain cells in the adult mammalian brain is limited, reptiles are able to regenerate large populations of neuronal cells. Pythons exhibit a characteristic specific dynamic action (SDA) response after food intake with an increase in metabolic rate that facilitates processing the meal. Associated with this change in SDA, pythons (Python spp.) also exhibit impressive plasticity in their digestive and cardiovascular physiology due to the sheer magnitude of the increase in organ growth that occurs after a meal to speed digestion, absorption, and assimilation of nutrients. While this systemic growth in response following food consumption is well documented, whether the python brain exhibits associated changes in cell proliferation following food consumption and digestion is currently unexplored. For this study, juvenile male ball pythons (Python regius) were used to test the hypothesis that postprandial neurogenesis is associated with food consumption. We used the thymidine analog 5-bromo-12’-deoxyuridine (BrdU) to quantify and compare cell proliferation in the brain of fasted snakes and at two time points: two days and six days after a meal, which span time periods of during and after SDA response, respectively. Quantification of BrdU-labeled cells in the ventricular regions relealed that – consistent with other reptile species – the retrobulbar and olfactory regions had the highest numbers of proliferating cells in the python brain, regardless of sampling time. Throughout the telencephalon, cell proliferation was significantly greater in the six-day post-feeding group, with no difference between the two-day post-feeding group and controls. Most other postprandial systemic plasticity occurs within a day or two after a meal and decreases thereafter; however, the brain displays a more delayed response, with a surge of cell proliferation after most of the digestion and absorption is complete. Our results support our hypothesis that food consumption does affect cell proliferation in the python brain, and indicates that the degree of increased proliferation is dependent on the time since feeding.

On the basis of a strong body of data, the Institute of Medicine currently recommends at least 60 minutes of exercise per day to prevent body weight gain overtime. Previous studies have shown that there is no compensatory increase in food intake with this dose of exercise. Ultimately, the brain decides whether to alter food intake. Surprisingly, no published studies have assessed the impact of exercise on brain activation. Using functional magnetic resonance imaging (fMRI) and an appetite questionnaire, we investigated the effects of a single bout of aerobic exercise on brain responses to visual food cues and subjective appetite responses. After an overnight fast, 30 (17M, 13W), healthy, habitually active subjects (22.0±3.8 years, 23.6±2.4 kg/m2, 44.3±8.3 mL∙kg-1∙min-1) either rested or exercised for 60 minutes, in a counterbalanced crossover design. Immediately after each condition, blood oxygen dependent levels were determined in response to visual food cues of different energy value during an fMRI scan. Exercise showed significantly greater activation (P < .005, uncorrected) in regions implicated in food inhibition (superior frontal gyrus, medial surface), and visual attention (precuneus, superior temporal gyrus, middle temporal gyrus and fusiform gyrus) regions. However, exercise did show a greater activation in a food reward region (medial orbitofrontal cortex). The rest condition only showed greater activation in a visual center (fusiform gyrus) and the midbrain. In addition, relative to no-exercise, subjective appetite responses were suppressed following the exercise bout. Taken altogether, these data suggest exercise may impact the brain in a direction expected to suppress food intake and increase food attention, which is in line with previous behavioral, biological and fMRI data. These findings may explain, at least partially, why aerobic exercise does not lead to a compensatory increase in food intake.

Anthradithiophene (ADT) derivatives have proven to be a front-runner in the world of small molecule semiconductors for organic electronics and optoelectronics. This is mainly due to the improved stability, easy tuning of chemical and physical properties, and impressive device performance that these molecules possess, especially in organic field effect transistors (OFET) and organic photovoltaics (OPV). The second chapter of this dissertation shows that reducing the amount of alkylsilylethynyl groups, used for functionalizing and solubilizing the ADT backbone, does alter the chemical, physical and crystallographic properties of ADTs. These changes offer the opportunity to study and observe different intermolecular interactions as well as monitoring their influence on sulfur scrambling in solid state. Additionally, from the early days ADTs and functionalized ADTs have been synthesized as isomeric mixtures. In chapter three, I demonstrate a new and simple method that can separate the syn and anti isomers of the F-TES-ADT and F-TEG-ADT chromatographically. The effects of isomeric purity on crystal packing and field effect transistor performance were studied extensively. Chapter four of this dissertation reveals a new generation of acceptor (electron poor) ADT derivatives obtained by attaching cyanide as electron withdrawing group (EWG) to the ADT chromophore. An extensive study was conducted on CN-ADT (acceptor) molecules in small molecule (F-TES-ADT) donor/ small molecule (CN-ADT) acceptor binary BHJ blends as well as P3HT/CN-ADT/PCBM ternary BHJ blends. Photophysical studies of the Donor/ acceptor blends (interface, domains, and crystal orientation) were conducted to obtain a better understanding of the film morphology and its effect on solar cell performance. Finally, the last part of the dissertation, Chapter five, focus on studying singlet fission in ADT derivatives, as well as the effect of varying the size of the alkylsilylethynyl functional group (used for solubilizing the ADT backbone) on altering the electronic couplings and how can that potentially affect the singlet fission rate in these molecules. We also tried to inspect the extent of the correlation between long-range order in crystal packing and singlet fission by monitoring singlet fission rate and efficiency for ADT derivatives with different thin film morphologies.

In recent years, the causal relationship between inflammation and cancer has gained wider acknowledgement and acceptance. While various types of immune cells are involved in the process of inflammation, macrophages represent the major inflammatory component of many tumors. Derived from circulating monocytes, these cells migrate to tumor sites in response to molecular cues present within the tumor microenvironment. Once there, interactions with neoplastic cells shape the differentiation and functional orientation of macrophages into two phenotypically distinct subsets: the “classically” activated M1 macrophages and the “alternatively” activated M2 macrophages. The preeminent paradigm in macrophage-related cancer research is that within the tumor stoma, macrophages acquire an M2 phenotype characterized by production of pro-angiogenic factors, ECM degrading enzymes and up-regulation of anti-inflammatory responses, thereby promoting tumor progression. M1 macrophages, on the other hand are thought to exert anti-tumorigenic effects due to their production of pro-inflammatory cytokines, and reactive oxygen species (ROS). While the generation of ROS during immune responses is an important aspect of immune regulation and host defense, excessive ROS production has been implicated in the pathogenesis of various degenerative diseases, including cancer. Yet, despite the well-established role of M1 macrophages in generating high levels of ROS via NADPH oxidase (NOX), M1 macrophages are still largely viewed as anti-tumorigenic. Hence, this study reevaluates the complex interaction between prostate cancer (PCa) cells and tumor-associated macrophages (TAMs), and operates on the premise that PCa cells promote a pro-tumor microenvironment, denoted by increased inflammation and oxidative stress, in part, through M1 macrophage-mediated, NOX-derived ROS production. Accordingly, immunofluorescent analysis of prostate tissue microarrays demonstrated an influx of M1 macrophages in prostate carcinoma. Immature monocytes co-cultured with the poorly tumorigenic prostate cell line, LNCaP, demonstrated changes in morphology and protein expression consistent with M1 macrophage polarization. PCa cells co-cultured with M1 macrophages displayed significantly higher intracellular ROS levels. Furthermore, M1-mediated ROS generation through NOXs increased prostate cell invasiveness and anchorage-independent growth. Taken together, results from this study suggest a potentially novel pro-tumorigenic function of M1 macrophages in early PCa progression, and aid in understanding the complex role of inflammation in cancer.

The field of prostate cancer research is in need of biological markers that predict which cancers do not need treatment, those that can be treated successfully with a localized treatment and more specific cases in which patients are likely to have an aggressive form of cancer that will require more aggressive surgical and chemotherapeutic treatments. ZIC2 is one of five members of a family of proteins that play critical roles in neural crest and mesoderm growth in normal embryonic brain development and in the adult cerebellum of vertebrates. Found throughout the animal kingdom, ZIC1-5 genes encode five distinct ZIC proteins containing five highly conserved C2H2-type zinc finger motifs whose structural integrity is important in carrying out its function as a transcription factor. We hypothesize that ZIC2 has functional significance at the molecular and cellular levels in the initiation of prostate adenocarcinoma (PRAD) and the progression to metastatic and/or castration resistant prostate cancer (CRPC). Bioinformatic predictions suggest that the function of ZIC2 is regulated by post-translational modifications, such as phosphorylation, ubiquitination and sumoylation. This proposal further outlines the research hypothesis for investigating the role of ZIC2 in prostate cancer progression and the effects of the post-translational modification, ubiquitination, on the loss or gain of function of ZIC2.

Transition-metal-catalyzed cross-coupling reactions are reliable tools for forging C–C bonds. The Engle Lab has previously pioneered the intermolecular difunctionalization of unactivated alkenes facilitated by nickel catalysis, where regioselectivity is controlled through the use of a bidentate directing group. A limitation of existing methods is that allyl groups have not yet been successfully incorporated, as the electrophile scope has been limited to alkyl and aryl species. Fundamentally, C–C p-bonds have served as key building blocks for the assembly of complex molecules, and the ability to introduce allyl moieties in a controlled manner enables diverse, downstream functionalization in multi-step synthesis. This work focuses on the use of diverse azaheterocycle directing groups connected to non-conjugated alkenes. Through the use of nickel catalysis, we have been able to successfully introduce and preserve allyl and cinnamyl species at the g-position and alkyl zinc nucleophiles at the b-position with high yield under mild conditions. This novel, 1,2-allylalkylation can accommodate a vast array of substituents with different electronic and steric properties (>20 examples). Our efforts have shifted to exploring different monodentate directing groups and to conduct mechanistic studies to shed light on the catalytic cycle. Interestingly, electron-rich electrophiles provide nearly quantitative NMR and isolated yields, whereas electron-poor electrophiles lead to lower yields. We report a competition experiment to further elucidate this mechanism. While isolated yields were generally higher for electron-rich groups, a competition between p-OMe and p-CF3 electrophiles led to preferential incorporation of the trifluoromethyl-substituted coupling partner, which supports oxidative addition as the product-determining step.