Ebola viruses cause sever disease in humans and non-human primates. The resulting disease, Ebola virus disease (EVD), can have hemorrhagic manifestations and has mortality rates ranging from 20-90%. There is a strong need for better understanding of the disease as well as improved diagnostics and prognostics. One approach to improving diagnostic and prognostics for severe viral diseases such as EVD is to define how the host response to infection develops and produces indicators of disease and outcome. To create a better means to understand if a patient is likely to survive or succumb to Ebola (EBOV) infection, I have sought to develop an understanding of the host response to EBOV infection in humans from the recent outbreak. I analyzed RNA-Seq samples from the 2013-2016 West Africa outbreak. I identified that the innate immune pathways are in general over activated in EVD and is stronger in patients who succumbed to disease. Furthermore, I developed a set of 10 genes that can perform as a prognostic indicator of disease independent of the viral load. This is the first demonstration that the circulating transcriptional immune response to EBOV infection can be used to predict infection outcome. To work towards a diagnostic platform of disease, I analyzed multiple studies of time-resolved datasets in animal models of disease. I analyzed RNA-Seq and NanoString data coupled with telemetry data in EBOV-challenged macaques. The earliest and strongest changes seen in the pre-symptomatic stage of disease is the up-regulation of many innate immune genes. I used this information to develop a NanoString codeset that can act as a pre-symptomatic indicator of disease that was tested in further animal studies as a diagnostic in pre-symptomatic stages of disease. Together, this work has identified a sets of genes that can work as a diagnostic for pre-symptomatic patients of EBOV and act as a prognostic indicators of disease. In future outbreaks, this type of information will be important to help track primary contacts of infected individuals and first responders, as well as better inform clinical management of patients. This lays the groundwork for similar analysis to be performed on other severe viral diseases such as Lassa Fever and Marburg Fever. / 2019-11-27T00:00:00Z

An earlier approach to restore homeostatic levels of ECM degrading matrix metalloproteases (MMPs) by the Sefton Lab utilized hydroxamate-based MMP inhibitory (MI) beads. While the MI beads delayed ECM degradation in the context of skin wound healing, they caused elevated cell infiltration in a subcutaneous implant model. The primary goal of this project was to further investigate this finding using an air pouch implant model in mice and a different control group – methacrylic acid-based (MAA) beads. Exudate analysis indicated that the MI beads, implanted subcutaneously with gelatin discs, elicited a similar biological response as the MAA beads. Exudates corresponding to both biomaterials had similar cell counts and chemokine levels, which were greater than those corresponding to the control used earlier, poly-methyl methacrylate-based (PMMA) beads. Further, both MI and MAA beads activated infiltrating macrophages in the classical manner, and influenced the activity of an MMP8 catalytic domain in a similar manner.

An earlier approach to restore homeostatic levels of ECM degrading matrix metalloproteases (MMPs) by the Sefton Lab utilized hydroxamate-based MMP inhibitory (MI) beads. While the MI beads delayed ECM degradation in the context of skin wound healing, they caused elevated cell infiltration in a subcutaneous implant model. The primary goal of this project was to further investigate this finding using an air pouch implant model in mice and a different control group – methacrylic acid-based (MAA) beads. Exudate analysis indicated that the MI beads, implanted subcutaneously with gelatin discs, elicited a similar biological response as the MAA beads. Exudates corresponding to both biomaterials had similar cell counts and chemokine levels, which were greater than those corresponding to the control used earlier, poly-methyl methacrylate-based (PMMA) beads. Further, both MI and MAA beads activated infiltrating macrophages in the classical manner, and influenced the activity of an MMP8 catalytic domain in a similar manner.

With the rising prevalence of diabetes, effective means of successful management of blood glucose levels are increasingly important. To improve on the ease of measurements, new technology is being developed to enable less invasive measurements. Some recent efforts have focused on the development of optical microscale glucose sensing systems based on the encapsulation of glucose oxidase within microspheres coated with polyelectrolyte multilayer nanofilms. In such sensors, a phosphorescent oxygen indicator is also co-encapsulated with the enzyme inside so that when glucose is present, glucose oxidase within the sensor reduces the local oxygen levels, causing a corresponding change in the luminescence intensity of the sensors. To test the aforementioned factors, a two-substrate, 2D FEM model of microscale optical glucose sensors in the dermis was developed. The model was used to predict the response time and sensitivity of glucose sensors with varying number and spacing of particles distributed in the dermis and varying physiological characteristics of the surrounding tissue; specifically, capillary density, blood vessel location relative to sensor, and glucose and oxygen consumption in tissue. Simulations were conducted to determine the magnitude of the change in the response time of sensors. Because the steady-state oxygen concentration within the sensors for a given blood glucose level determines the signal output, steady-state concentration of oxygen within sensors and the surrounding tissue for the entire physiological glucose range was evaluated. The utility of the model to predict the performance and efficacy of the sensors in the event of a host response to the foreign body implant was also evaluated. Simulations were performed to evaluate changes in sensor response and sensitivity in the occurrence of inflammation and progression of fibrous encapsulation of various thickness and density. The results from these simulations have provided knowledge on the impact of physiological factors that can potentially degrade sensor function in vivo. Our results indicate that upon the occurrence of a host response, sensitivity is reduced while range is extended. Furthermore, using the model we have been able to determine which conditions in vivo improve response time, sensitivity, and the linear response range for these sensors.

Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF-α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles.

Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF-α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles.

Toxoplasma gondii is the causative agent of Toxoplasmosis in human and animals. T. gondii isolates are highly diverse. Hundreds of genotypes have been identified, but only three clonal lineages, namely Type I, II and III are prevalent worldwide. In mouse model, T. gondii strains can be divided into three groups based on their virulence, including the virulent (LD100=1), the intermediately virulent (LD50 = 103-104) and the non virulent (LD50 > 105). The clonal Type I, II and III T. gondii strains belong to these three groups, respectively. Epidemiologic studies suggest the difference of virulence in mice may relate to the severity of toxoplasmosis in human infection. Therefore, it is necessary to understand biological differences in genetically different T. gondii strains and their effect on the host responses. To date, the majority of data published on this aspect has been limited to in vitro assays. Here, we used in vivo assays to investigate host responses upon infection of distinct Toxoplasma strains. Our studies examined host response to infection of the three widespread clonal lineages of T. gondii using a mouse model. The following results were revealed: (i) increased tissue burden in mice is the indicator of virulence of T. gondii. Quantification of parasite burden in the spleen of mice showed significantly more parasites for Type I strain than that of Type II and III strains, with the latter two having comparable parasite burdens. Given that the Type II strains are more virulent than the Type III strains in mice; this result suggests that difference in host response is the result of specific parasite-host interaction, which is not simply due to the difference of parasite tissue load. (ii) gene expression in the host is strongly influenced by parasite genetic background. Transcriptional profiles of mice infected with the above three types of T. gondii strains showed that the overall gene expression patterns are similar between Type I and Type II infected mice and both stimulated stronger and more polarized change comparing to Type III strain. These results emphasize the importance of studying T. gondii pathogenesis in the host with the consideration of parasite genetic diversity. Such research could possibly aid in select appropriate regimes to treat toxoplasmosis caused by diverse T. gondii strains.

Neural electrodes are an important part of brain-machine interface devices that can restore functionality to patients with sensory and movement impairments including spinal cord injury and limb loss. Currently, chronically implanted neural electrodes induce an unfavorable tissue response which includes inflammation, scar formation, and neuronal cell death, eventually causing loss of electrode functionality in the long term. The objective of this research was to develop a coating to improve the tissue response to implanted neural electrodes. The hypothesis was that coating the surface of neural electrodes with a non-fouling, anti-inflammatory coating would cause reduced inflammation and a better tissue response to the implanted electrode. We developed a polymer coating with non-fouling characteristics, incorporated an anti-inflammatory agent, and engineered a stimulus-responsive degradable portion for on-demand release of the anti-inflammatory agent in response to inflammatory stimuli. We characterized the coating using XPS and ellipsometry, and analyzed cell adhesion, cell spreading, and cytokine release in vitro. We analyzed the in vivo tissue response using immunohistochemistry and microarray qRT-PCR. Although no differences were observed among the samples for inflammatory cell markers, lower IgG penetration into the tissue around PEG + IL-1Ra coated electrodes suggests an improvement in BBB integrity. Gene expression analysis showed higher expression of IL-6 and MMP-2 around PEG + IL-1Ra samples, as well as an increase in CNTF expression, an important marker for neuronal survival. An important finding from this research is the increased neuronal survival around coated electrodes compared to uncoated controls, which is a significant finding as neuronal survival near the implant interface is an essential part of maintaining electrode functionality.

As the Eucalyptus globulus plantation industry expands and matures in southwestern Australia (WA), the impact of disease within the plantation environment is predicted to increase. This thesis investigated the most abundant canker-causing pathogen associated with branch and stem cankers, Endothiella eucalypti the anamorph of Cryphonectria eucalypti. Endothiella eucalypti was widespread, although at low incidence, throughout the WA plantation estate and was frequently observed sporulating on the bark of healthy hosts in the absence of disease. Regions with a long (approximately 20 years) plantation history, such as Bunbury, had the highest incidence of this pathogen. A high degree of variability in pathogenicity, growth rate and colony morphology was observed between WA isolates of En. eucalypti. In the glasshouse, a significant variation in susceptibility of seven E. globulus provenances to En. eucalypti was observed. Although an interaction between the E. globulus provenance and En. eucalypti isolate was recorded, some provenances were generally more susceptible than others. In two 18-month-old plantations, the susceptibility of three provenances to En. eucalypti was significantly influenced by environmental conditions. Visual assessment of general tree health indicated that less healthy trees had smaller lesions than healthy trees. It is hypothesised that the selection of E. globulus provenances to suit site conditions in the future should decrease the risk of serious disease, especially on marginal sites. Endothiella eucalypti caused disease in intact stems of two-year-old E. globulus under glasshouse conditions. This suggests that En. eucalypti may not require a wound to infect in the field. Vegetative compatibility groupings between WA En. eucalypti isolates indicated a relatively high degree of genotypic diversity within the WA asexual population of En. eucalypti, whereas inter-simple sequence repeats PCR (ISSR-PCR) analysis indicated a lower level of genotypic diversity. Discrepancies between traditional and molecular techniques, such as ISSR-PCR, was attributed to the more specific gene-togene analysis afforded by molecular techniques. ISSR-PCR successfully distinguished variability within the En. eucalypti population and with the teleomorph, isolated in South Africa. It also separated Cryphonectria cubensis isolates from the C. eucalypti isolates. As copper is the micronutrient most limiting growth of E. globulus in WA, its role in the resistance of two E. globulus provenances was examined in a glasshouse trial. Lesion extension or defence responses of E. globulus to En. eucalypti did not differ between Cu-adequate and Cu-deficient plants. It is suggested that constitutive levels of host defence enzymes played a more important role in providing protection for the host against En. eucalypti than the external supply of copper. A reduction in the canopy volume of E. globulus within plantations due to insect herbivory or foliar pathogens, such as Mycosphaerella spp., has been reported to predispose the host to disease caused by non-aggressive canker-causing fungi. Under two separate glasshouse trials, conditions of 100% defoliation and 80% defoliation maintained over six weeks prior to inoculation, were required to significantly increase lesion extension caused by En. eucalypti in E. globulus stems. The ability of defoliated E. globulus to retain a degree of resistance to En. eucalypti was attributed to the rapid replacement of foliage and up-regulation of photosynthesis in remaining leaves. The carbohydrate reserves of the plant were depleted following defoliation and remained depressed regardless of the length of time the trees remained defoliated. In conclusion, the endophytic habit of En. eucalypti poses a threat to highly stressed trees, however it does not appear to be an immediate threat to WA plantation health. Although En. eucalypti has not yet been responsible for a major disease outbreak in WA, the impact of this disease on plantation-grown eucalypts elsewhere in Australia and worldwide serves as an indication of its potential to affect WA plantations.

<I>Orobanche</I> (spp.) are parasitic plants that attack the roots of many important crops. <I>O. aegyptiaca</I> penetrates the host root (aided by digestive enzymes) and forms connections to the host vascular tissue, from which it will withdraw all of its water and nutrient requirements. In order to control this weed, it is important to understand the relationship between the host and the parasite. To investigate how parasitism effects host defense pathways, we are studying the patterns of expression of host genes known to be involved in various aspects of plant defense responses. With respect to local defense responses, two genes of the isoprenoid pathway were studied, one of which is expressed in wounded tissue (<I>hmg1</I>), and another that is induced in response to wounding yet repressed in response to pathogen elicitors (squalene synthase). Genes analyzed that are associated with systemic defense include <I>PR-1</I>, <I>PR-2</I>, and <I>PR-5</I>, all of which are induced in response to pathogen attack as part of the systemic acquired resistance (SAR) response. Plant gene expression was studied using transgenic tomato plants containing <I>hmg1</I>-GUS fusions, and northern hybridization analysis of tobacco and Arabidopsis roots using gene-specific probes. Results indicated that expression of <I>hmg1</I> is induced, <I>PR-2</I> and squalene synthase are repressed, and <I>PR-1a</I>, <I>PR-1</I>, and <I>PR-5</I> are not affected in tissue parasitized by <I>O. aegyptiaca</I>. Together, these results indicate a complex response to the parasite. Whereas <I>hmg1</I> induction is consistent with <I>O. aegyptiaca</I> inflicting a simple wound-like injury, the repression of squalene synthase is consistent with plant recognition of a pathogen attack. In contrast, the failure of <I>Orobanche</I> to induce SAR- related <I>PR-1</I> in tobacco and <I>PR-1</I>, <I>PR-2</I>, or <I>PR-5</I> in Arabidopsis indicates an ability to avoid or perhaps inhibit some defense-related pathways. By comparing the regulation of these defense genes in response to <I>O. aegyptiaca</I> attack, we are able to gain a greater understanding of the host plant response to parasitization and explore potential gene candidates for future engineering strategies to create <I>Orobanche</I> resistant crops. / Master of Science