Cognitive Impairment in Mild Traumatic Brain Injury| A Diffusional Kurtosis and Perfusion Imaging Study

<p> In addition to being the natural genetic information carrier, DNA can also serve as a versatile material for construction of nanoscale objects. By using the base-pairing properties of DNA, we have been able to mass-produce nano-scale structures in a variety of different shapes, upon which patterns of other molecules can be further specified. The diversity of molecules and materials that can be attached to DNA and the capability of providing precise spatial positioning considerably enhance the attractiveness of DNA for nano-scale construction. A further challenge remains to use these DNA based structures for biomedical applications. </p><p> As proof-of-concept, a DNA-based nanodevice for multivalent thrombolytic delivery is designed, which intends to employ DNA nanostructures as carriers for the delivery of tissue plasminogen activator (tPA) and plasminogen. Universal modular adapter molecules that can simultaneously bind "down" to the DNA structures and "up" to these thrombolytic drugs are further proposed. We begin with exploring the molecular recognition properties provided by biotin-avidin and aptamer-ligand pairs, and are able to achieve site-specific display of certain protein targets along the DNA nanostructure scaffold. Yet for both of these approaches, only biotinylated or specially selected proteins can be patterned. We further propose to develop single-chain diabodies (scDb) as the adapter molecules. This scDb approach is highly modular and can be extended to assemble virtually any proteins and therapeutic molecules of interests, which at the same time will greatly enhance our molecular toolbox for nanoscale construction.</p> / Dissertation

<p>Thymus atrophy is highly inducible by stress and prolonged thymus atrophy can contribute to T cell deficiency or inhibit immune recovery after acute peripheral T cell depletion. Little is known regarding the mechanisms driving thymic involution or thymic reconstitution after acute stress. Leptin deficiency in mice results in chronic thymic atrophy, suppressed cell-mediated immunity, and decreased numbers of total lymphocytes, suggesting a role for leptin in regulating thymopoiesis and overall immune homeostasis. Exogenous leptin administration during stress has been shown to protect against thymic damage, yet the mechanisms governing these thymostimulatory effects are currently undefined. Studies herein define the impact of endotoxin-induced thymic damage in the stromal and lymphoid compartment of the thymus and systemic glucocorticoid and cytokine responses in the animal. We report here the novel finding that leptin receptor expression is restricted to medullary thymic epithelial cells in the normal thymus. Using a model of endotoxin-induced acute thymic involution and recovery, we have demonstrated a role for the metabolic hormone leptin in protection of medullary thymic epithelial cells from acute endotoxin-induced damage. We also demonstrated that systemic leptin treatment decreased endotoxin-induced apoptosis of double positive thymocytes and promoted proliferation of double negative thymocytes in vivo through a leptin receptor isoform b-specific mechanism. Leptin treatment increased thymic expression of IL-7, an important soluble thymocyte growth factor produced by medullary thymic epithelial cells. We also found leptin to inhibit systemic glucocorticoid and pro-inflammatory cytokine responses. Using leptin-deficient and leptin receptor-deficient mice in our stress model, we found that endotoxin-induced thymic atrophy was exacerbated in the absence of leptin, despite an inability to mount a proper pro-inflammatory cytokine response. Together, these data support a model in which leptin can function to protect the thymus gland from stress-induced acute damage in part by reduction of systemic corticosteroid and pro-inflammatory cytokine responses, and intrathymically through a mechanism orchestrated by medullary thymic epithelial cells and their soluble mediators (e.g. IL-7). Taken together, these studies suggest a physiological role for leptin signaling in the thymus for maintaining healthy thymic epithelium and promoting thymopoiesis, which is revealed when thymus homeostasis is perturbed by stress.</p> / Dissertation

<p><p>The receptor-recognized form of &alpha;₂-macroglobulin (&alpha;₂M*) targets antigens (Ag) to professional Ag-presenting cells (APCs) for rapid internalization, processing, and presentation. When employed as an Ag delivery vehicle, &alpha;₂M* amplifies major histocompatibility complex (MHC) class II presentation as demonstrated by increased antibody (Ab) titers. Recent evidence, however, suggests that &alpha;₂M*-encapsulation may also enhance Ag-specific cytotoxic T lymphocyte (CTL) immunity. In these studies, we demonstrate that &alpha;₂M*-delivered Ag (ovalbumin, OVA) enhances the production of specific <italic>in vitro</italic> and <italic>in vivo</italic> CTL responses. <br><p>Murine splenocytes expressing a transgenic T cell receptor (TCR) specific for CTL peptide OVA_257-264 (SIINFEKL) demonstrated up to 25-fold greater IFN-&gamma; and IL-2 secretion when treated <italic>in vitro</italic> with &alpha;₂M*-OVA compared to soluble OVA. The frequency of IFN-&gamma; -producing cells was increased ~15-fold as measured by ELISPOT. Expansion of the OVA-specific CD8<super>+</super> T cells, as assayed by tetramer binding and [<super>3</super>H]thymidine incorporation, and cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also significantly enhanced by &alpha;₂M*-OVA. Furthermore, CTL responses were observed at Ag doses tenfold lower than those required with OVA alone. <br><p>We also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with &alpha;₂M*-OVA. These &alpha;₂M*-OVA-immunized mice displayed increased protection against a subcutaneously implanted OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The anti-tumor response observed with &alpha;₂M*-mediated Ag delivery was comparable to that of an accepted vaccine adjuvant (CpG 1826) and appeared superior to a cell-based vaccine technique. <br><p>To further understand the mechanism underlying this enhanced CTL immunity, the subsets of professional APCs capable of cross-presenting &alpha;₂M*-encapsulated Ag were investigated. Although both dendritic cells (DCs) and macrophages appear to stimulate some degree of cross-priming in response to &alpha;₂M*-encapsulated Ag, CD8<super>+</super>CD4<super>-</super> and CD8<super>-</super>CD4<super>+</super> DCs appear to do so with the greatest efficiency. The implications of this finding to the ongoing debate regarding the relative contributions of APC subsets to Ag cross-presentation and the determinants of which cells cross-present with high efficiency are discussed. <br><p>These observations demonstrate that &alpha;₂M*-mediated Ag delivery promotes cross-presentation resulting in enhanced Ag-specific CTL immunity. Considered in the context of previous work, these results support &alpha;₂M* as an effective Ag delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.</p> / Dissertation

<p>The liver is a primary target for toxicants and/or their metabolites. Selected fish species now serve as model organisms for laboratory investigations of toxic responses in the liver. One such model is the Japanese medaka (<italic>Oryzias latipes</italic>), a small freshwater teleost with a robust history of usage in liver and biliary toxicity studies. The structural components of the medaka hepatobiliary system have been well-described by recent studies in two- and three-dimensional contexts, but efforts to characterize the molecular mechanisms underlying critical medaka liver functions during normalcy remain sparse. This dearth of information makes it difficult to definitively characterize toxic responses in this model organism. A crucial transcription factor underlying proper hepatobiliary function in both mammalian and non-mammalian species is the farnesoid X receptor alpha (FXR&alpha;), a member of the nuclear receptor superfamily that plays a key role in bile acid homeostasis. This dissertation describes the function of medaka fxr&alpha; during both normalcy and toxicity.</p><p>To achieve this overall objective, <italic>in vitro</italic> techniques were first employed to study the function of medaka <italic>fxr&alpha;</italic>. Two isoforms of <italic>fxr&alpha;</italic> that differ in the AF1 domain, Fxr&alpha;1 and Fxr&alpha;2, were isolated from liver cDNA and are the result of alternative splicing of one gene locus. Fxr&alpha;2 responded significantly to C24 bile acids and the synthetic FXR&alpha; agonist GW4064. On the other hand, Fxr&alpha;1, despite having an identical ligand-binding domain to that of Fxr&alpha;2, showed no response to any agonists tested by transient transactivation assays. Furthermore, Fxr&alpha;2 interacted with nuclear receptor coactivators PGC-1&alpha; and SRC-1 in mammalian two-hybrid assays while Fxr&alpha;1 did not. These findings point to a significant importance of the AF1 domain to overall receptor structure and function. </p><p>Following <italic>in vitro</italic> functional characterization, <italic>in vivo</italic> experiments using medaka larvae were performed to determine <italic>fxr&alpha;'s</italic> function during normalcy. Quantitative, real-time PCR data demonstrated that Fxr&alpha;1 is highly expressed in adult liver, while Fxr&alpha;2 is expressed predominantly in gut. Fxr&alpha;1's expression was higher than Fxr&alpha;2 in embryos and larvae at all developmental timepoints tested. In vivo exposures of medaka hatchlings to GW4064 at various doses significantly altered expression of defined FXR&alpha; targets, including: bile salt export protein (BSEP), small heterodimer partner (SHP), and cytochrome P450 7A1 (CYP7A1). Surprisingly, numerous sublethal hepatic alterations to hepatocytes and bile preductular epithelial cells (BPDECs) were observed following exposure to GW4064; alterations included: lipid accumulation, glycogen depletion, mitochondrial swelling and rupture of mitochondrial membranes, disruption of endoplasmic reticulum, and apoptosis. Significant lipid accumulation, as revealed by oil red O whole mount staining of larvae, was also noted at lower doses of GW4064. These findings were the first observations of sublethal hepatotoxicity of GW4064; to date, no studies in the mammalian literature reported alterations following its administration.</p><p>Because of studies in the mammalian literature that demonstrated alleviation of cholestatic injury induced via the classic hepatotoxicant &alpha;-naphthylisothiocyanate (ANIT) by GW4064, it was originally hypothesized that a similar finding would be observed in medaka coexposed to these compounds. However, because of GW4064's ability to induce sublethal hepatic alterations in medaka, it was anticipated that its coadministration with ANIT would result in enhanced toxicity rather than alleviation as described in rodent models. However, despite the sublethal alterations induced by 1 uM GW4064, alleviation of toxicity following exposure to 15 uM ANIT was observed. Surprisingly, reduction of GW4064's toxicity was also observed in larvae exposed to both compounds. These investigations of <italic>fxr&alpha;</italic> function are an important and essential component in furthering our understanding of hepatobiliary toxicity in small aquarium fish models of human liver disease. These collective findings have created molecular underpinnings necessary for understanding medaka hepatobiliary function during normalcy and toxicity.</p> / Dissertation

<p>Sepsis is a major cause of morbidity and mortality in the United States, and Staphylococcus aureus (S. aureus) is the bacteria most commonly cultured from septic patients. In severe sepsis, the relationship between the systemic inflammatory response and the resulting mitochondrial and metabolic dysfunction is not fully understood, especially with respect to the mechanisms of mitochondrial damage resolution. The process of mitochondrial biogenesis, which leads to the restoration of metabolic and anti-oxidative functions in damaged or stressed cells and tissues, is pro-survival and is a critical protective response in sepsis. Mitochondrial biogenesis requires the coordinated expression of multiple regulatory proteins, including the PPARgamma-coactivator (PGC) family of proteins. Previous work in sepsis has focused on mitochondrial biogenesis in response to late signals of mitochondrial damage; however, for acute sepsis, we have hypothesized a direct and early link between the innate immune response and the transcriptional activation of mitochondrial biogenesis. Since the Toll-like receptors (TLRs) are a major part of the innate immune response, we hypothesized that they could activate mitochondrial biogenesis in bacterial sepsis. Earlier work showed that TLR4 (which responds to components of Gram-negative bacteria) was necessary for mitochondrial biogenesis induction in response to heat-killed E. coli challenge. For this work, the objective was to investigate whether signaling by TLR2 (which responds to components of Gram-positive bacteria) would activate mitochondrial biogenesis in response to S. aureus sepsis in mice. The sepsis model was initially characterized in wild-type (WT) mice by PCR analysis of hepatic RNA, in which the up-regulation of several regulatory proteins for mitochondrial biogenesis, including all three PGC family members, was observed. In contrast, in both TLR2-/- and TLR4-/- mice, the mitochondrial biogenesis response was deficient and delayed. In addition, PGC-1alpha and PGC-1beta were differentially regulated in WT, TLR2-/-, and TLR4-/- mice. To identify the mechanisms involved in this induction pattern, the known TLR signaling pathways were systematically probed for activation using several strains of genetic knockout mice. These data demonstrated that the differential regulation of the PGC family is independent of the MyD88 adapter protein and is caused in part by IRF7 signaling. IRF7 is a pro-inflammatory transcription factor that is normally involved in the interferon response; in this case, IRF7 was found to be necessary but not sufficient for PGC-1alpha/beta induction. In addition, a second level of regulation was identified in the microRNA mmu-mir-202-3p, which is inversely correlated with the expression of PGC-1alpha and PGC-1beta mRNA in WT, TLR2-/-, and TLR4-/- mice and was shown to functionally decrease PGC-1alpha mRNA. If these observations are confirmed in humans, IRF7 and mir-202-3p may be potential therapeutic targets for the up-regulation of PGC-1alpha/beta levels in the clinical setting of sepsis and impaired mitochondrial biogenesis.</p> / Dissertation

Vertical transmission of HIV-1 represents a major, global health concern with particular regard to developing areas of the world, and perinatally acquired infections account for the majority of all pediatric HIV-1 cases. Maternal-fetal transmission of HIV-1 occurs at three stages: prepartum, intrapartum, and post-partum, however the mechanism of this transmission is not yet clearly defined. Additionally, the rate of transmission is estimated at ∼30%, leaving ∼70% of infected mothers who do not transmit to their infants. While several maternal factors including viral load, clinical stage of the mother, low CD4 counts, recent infection, and the maternal immune response to infection have been implicated in transmission, the viral factors which may influence transmission are not known. In this study, we have investigated the molecular and biological properties associated with the env V3 region and the entire env from both transmitting and non-transmitting mothers. Our results show that the minor genotypes of the mothers' heterogeneous viral populations are transmitted to their infants, the biological properties associated with the transmitted variants' V3 regions appear to be macrophage tropic (R5) and non-syncytium inducing, and the transmitted variants are initially maintained in the infants with the same properties. In addition, the molecular analyses of the env of non-transmitting mothers, including the variable regions V1-V2, V3, and V4-V5 were found to be significantly more homogeneous than that of transmitting mothers, suggesting that a limited heterogeneity within an infected mother may prevent vertical transmission. Since effective therapies should target the specific properties of transmitted variants, these results provide significant insight into the molecular mechanisms of maternal-fetal transmission, aiding the development of effective therapies for prevention of transmission and treatment of disease.

Cryptosporidiosis is a major cause of diarrheal illness worldwide and characterized by several daily bowel movements, resulting in fluid loss and dehydration. Two species, Cryptosporidium hominis and C. parvum are the main causative agents in human infection. Complicating matters for disinfection, epidemiology, and treatment studies, C. parvum isolates infect multiple mammalian species while C. hominis solely infects humans. The purpose of this dissertation was to: (1) develop a C. parvum PCR based detection method and discuss its limitations; and (2) to extend current epidemiological and molecular data rationalizing the multiple C. parvum host specific infectivity patterns. To fulfill these two objectives, three separate experiments were designed and executed. The results from which are included in the appendix as peer reviewed published manuscripts and are the basis of this dissertation. The first manuscript outlines the use and validation of microscopic micromanipulation to isolate and deliver low numbers of C. parvum oocysts to a test vial of interest. In addition, a nested PCR primer set was developed targeting the 18S rRNA and tested for sensitivity using micromanipulation and specificity using isolated DNA from multiple different species. It was determined that micromanipulation is an accurate technique able to deliver low numbers of oocysts to a test vial of interest. The nested PCR protocol had LLOD, in replicates of 50 and laboratory grade water, of 100% with ten oocysts and 38% with a single oocyst. The second manuscript compared detection efficiencies of the EPA Method 1623 with the nested PCR protocol outlined in the first manuscript. Both methods had equal detection efficiencies giving positive detection at the five-oocyst level. In addition, non-specific PCR amplification results generated during the study revealed specificity issues that have implications effecting past, current and future molecular detection validation processes. The final manuscript describes nucleotide and deduced amino acid differences between the C. parvum and C. hominis attachment/invasion surface proteins Cpgp 40/15, p23, and GP900. This information has implications explaining host-specificity differences observed among Cryptosporidium spp.

Clostridium difficile-associated disease (CDAD) in neonatal pigs has emerged as a serious economic concern for swine producers throughout North America. The disease has been diagnosed clinically and reproduced in experimental inoculation trials in pigs, but little is known of the epidemiology or pathogenesis of the disease in pigs. Strain characteristics and distribution of C. difficile isolates from pigs, calves, dogs, horses, and humans were assessed by PCR-ribotyping. Porcine and bovine isolates were dominated by a single ribotype. This ribotype was uncommon among isolates from other host species; it was particularly uncommon from humans, suggesting there is little transfer of isolates between humans and calves or pigs. The reason for a single common ribotype circulating among distinct bovine and porcine populations is unknown. The intragastric inoculation of newborn pigs demonstrated their sensitivity to C. diffcile toxins. Toxin B (TcdB) surprisingly resulted in more severe lesions than Toxin A (TcdA). The two toxins together acted synergistically. Colon explants were sensitive to TcdA in a dose-dependent manner. However, TcdB did not cause significant lesions in the explants, nor was there any synergism with TcdA. Electron microscopy of colon explants treated with TcdA revealed severe, ultrastructural lesions that accrued in a dose-dependent manner by two h post infection. Direct immunohistochemistry assays demonstrated specific binding of biotinylated TcdA throughout the gastrointestinal tract of neonatal pigs. The density of bound toxin in different segments correlated with the severity of lesions in those segments from pigs gavaged with TcdA. TcdB did not bind any tissues, though it was fully active in cell-culture assays. A monoclonal antibody to Galalpha1-3beta1-4GlcNAc-R (alpha-Gal epitope), a putative receptor for TcdA in pigs, specifically bound the brush border of enterocytes, but the distribution of binding did not correlate with the distribution of TcdA binding. Specific TcdA binding to the plasmallema of microvilli was also confirmed by immunoelectron microscopy. By five min post inoculation some toxin was already visible in endosomes or free in the cytoplasm. TcdA localized to the mitochondria of epithelial cells and, less frequently, to the nuclei. Endothelial cells and leucocytes in the superficial lamina propria were similarly labeled by toxin.

The consensus view is that cardiac hypertrophy is an adaptive response to increased work caused by a variety of stimuli. While hypertrophy can be defined as an increase in cell mass without an increase in cell number, not all increases are equivalent in type and amount of protein accumulated. Our goal in this study was to identify the common steps in the process of cardiac hypertrophy. Our working hypothesis was that in all forms of cardiac hypertrophy glucose utilization increases and that the percentage of energy derived from fatty-acid oxidation decreases. The first part of this study entailed the development and characterization of a neonatal rat heart cell model. The model had to provide uniform culture conditions for rapid development of hypertrophy by agents acting at different sites in the cardiomyocytes. The second part of this study was composed of an assessment of hypertrophy caused by four pharmacologically distinct agents: norepinephrine, angiotensin-II, endothelin-I and tetradecylglycidic acid. In this part we compared the quantity of protein accumulation and quality of hypertrophy cause by each agent. This task was accomplished by examining the effect of each agent on selected mRNA messages and alteration in DNA content of cardiomyocytes. Here we also examined the effect of protein kinase-C, endothelin-I and angiotensin-II inhibitors on hypertrophy caused by each agent. In the final part of this study, metabolic alteration in hypertrophy caused by each agent was assessed for a potential common pathway to hypertrophy. As part of this analysis, we examined changes in glucose and palmitate oxidation, glucose uptake and role of pentose pathway in hypertrophy resulting from treatment of cardiomyocyte by each agent.