The enormous mortality burden exerted by P. falciparum malaria has evolutionarily selected for red blood cell (RBC) polymorphisms which confer protection against the severe manifestations of this disease. Although the epidemiological protection by these polymorphisms has been well-established for the past half-century, the mechanisms underlying this protection are still being uncovered. Recent studies implicate impaired cytoadherence to microvascular endothelial cells (MVECs) due to reduced surface levels and altered display of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as a mechanism of protection against severe malaria by sickle hemoglobin (Hb) S and HbC. Consequently, in this thesis, I have described three separate, but related investigations into whether hemoglobins S and C influence a parasite’s cytoadherence binding phenotype (Chapter 3), the PfEMP1 variants that parasites express in vivo (Chapter 4), and the IgG recognition of PfEMP1 domains in Malian children (Chapter 5). We found that parasites from HbAS children show statistically insignificant increased binding to MVECs and that parasites did not express a restricted subset of var genes in HbAS and HbAC children. Compared to HbAA and HbAC children, HbAS children demonstrated a slower rate of acquisition of IgG responses to a repertoire of PfEMP1 domains. These findings suggest that, although hemoglobin type influences the binding phenotype of P. falciparum isolates and the acquisition of PfEMP1-specific IgG responses, other factors more likely determine the expressed var gene repertoire within parasites than hemoglobin type.

In the search for a vaccine providing comprehensive protection against meningococcal disease, one vaccine currently under development contains the immunogenic proteins PorA and FetA in meningococcal outer membrane vesicles (OMVs). To achieve high levels of coverage against disease-causing isolates, the antigenic variability of these proteins could be overcome using knowledge of meningococcal epidemiology and population structure. In this study, the possible implications of variable expression levels of PorA and FetA on vaccine efficacy were investigated. Producing OMVs containing consistent amounts of FetA is difficult due to iron-repressed expression; therefore, meningococcal strains were constructed which constitutively expressed FetA at increased levels for OMV vaccine production and analysis. In mice, OMVs from modified strains induced antibodies against both PorA and FetA. These antibodies acted synergistically in a serum bactericidal assay; however, antibodies against FetA were weakly bactericidal alone. The potential to increase levels of PorA- and FetA-specific bactericidal antibodies with a prime-boost strategy, using OMV and protein inoculums, was also tested. While successful for a weakly-immunogenic PorA variant, a similar strategy did not increase bactericidal activity against FetA. Although antibodies against FetA can be induced following OMV immunisation, sufficient antigen expression in target bacteria is also required for bactericidal killing; therefore, the variability and regulation of porA and fetA transcription was investigated in a range of isolates. Despite differences in regulation among clonal complexes, variable expression is unlikely to be an issue for vaccine coverage. In particular, regulation of fetA by iron is reduced in many isolates due to a deletion in the sequence bound by the regulatory protein, Fur. Therefore, a vaccine targeting PorA and FetA may provide high levels of protection against meningococcal disease; however, an alternative formulation or immunisation strategy is required to improve coverage against FetA.

Sialic acid (SA) is a known major virulence factor of Haemophilus influenza (Hi). This study aims to analyse the functions of some candidate sialometabolism genes, and to further our current understanding on the Hi sialometabolism gene regulation. Two candidate sialometabolism genes (HI0227 and HI0148) and their adjacent ORFs (HI0228, HI0148.1 and HI0149) were studied. HI0148.1 and HI0149 are transcribed as a single gene in screened NTHi strains, and we refer to the combined ORF as NTHI0236 (the designation in strain 86-028NP). Across Hi strains screened, the sequences of HI0227, HI0148 and NTHI0236 are conserved. However, the sequence of HI0228 is heterogeneous. Mutants that lack the functions of HI0227, HI0228, HI0148 and NTHI0236 were compared to their respective wild type parent strains for ability to grow on SA (in aerobic and microaerophilic conditions), their ability to sialylate LPS and their ability to resist complement mediated killing. The mutants did not exhibit major differences in the tested aspects of sialometabolism compared to their respective wild type strains. Changes observed in some of the mutants in serum bactericidal assays and LPS profiles were due to the effect of phase variable genes. The sialometabolism functions of HI0227, HI0148, and NTHI0236 remain obscure, and we postulate that HI0228 is a pseudogene. Investigation of Hi sialometabolism gene regulation was conducted using mutants that lack different steps of the Neu5Ac catabolism pathway and the Neu5Ac activation pathway. The expression of nanE and siaP, respectively representing the Neu5Ac catabolism and transport operons, were assessed using RT-PCR and qPCR. We investigated a temporal/concentration effect of Neu5Ac on the expression of sialometabolism operons, which highlights the importance of studying the Hi sialometabolism gene regulation as a dynamic process. We further demonstrated that GlcN-6P, a Neu5Ac intermediate from the catabolism pathway, is likely the SIS sugar that interacts with SiaR, the repressor protein of the Hi sialometabolism operons. We postulate that upon binding of GlcN-6P to SiaR, the SiaR-mediated repression on the Hi sialometabolism operons is relieved, resulting in the induction of the expression of Neu5Ac catabolism and transport genes.

Lower respiratory tract infections (LRTIs) are one of the most common infectious diseases in Europe and worldwide. Very little is known about the genetic factors associated with susceptibility to LRTI and so far studies have only analysed candidate gene loci. This work aimed to find genetic loci associated with susceptibility to severe and mild LRTI. To analyse severe LRTI, a European cohort of community acquired pneumonia (CAP) sepsis patients was used. To analyse mild LRTI, patients attending primary care with a cough were recruited across Europe. Two important candidate genes for susceptibility to severe and mild LRTI were studied. None of the four functional polymorphisms analysed in MBL2 were associated individually or when combined with susceptibility to LRTI or survival from severe LRTI. Rare homozygotes for rs12252 in the IFITM3 gene were found to associate with susceptibility to mild and severe viral LRTI, particularly influenza infection. Using a combination of genome-wide and candidate gene methods, 93 single nucleotide polymorphisms (SNPs) were chosen to genotype and analyse for susceptibility to mild LRTI. A gene region containing the MPHOSPH8, TPTE2, PSPC1 and ZMYM5 genes was found to associate with susceptibility to rhinovirus infection. A functional promoter SNP in the IL6 gene and a gene region containing the SAP18, MRP63 and SKA3 genes were associated with susceptibility to mild LRTI in smokers. A GWAS was performed for susceptibility to CAP sepsis in Europe. Genotypes were imputed for eight GWAS data sets comprising over 1000 CAP sepsis cases and over 4000 controls. Preliminary analysis results suggest an association in a chromosome 1 region containing the genes VASH2, ANGEL2 and RPS6KC1. This thesis presents results of the largest known genetic association study for susceptibility to CAP sepsis, and the only known genetic association study for mild LRTI. These novel findings will facilitate further research which may lead to better treatment and outcome for LRTI.

Mastitis represents a major issue within the dairy industry responsible for economic loss via decreased animal productivity and associated veterinary costs. Currently, there is a push to identify a phenotypic innate immune response that will yield dairy cows with an enhanced resistance to mastitis. Bovine dermal fibroblasts were used as a cell model to measure the response of individuals to Gram-negative bacterial stimuli through the TLR4 signaling pathway. Fibroblast cultures were isolated from 15 dairy heifers at 5, 11, and 16 months of age in order to determine the variability in responsiveness to LPS as well as to monitor the development of the innate immune response in calves. These individuals displayed a large range in response to LPS as measured by IL-8 production. In addition, response within individuals increased dramatically with age. To determine the cause behind this, DNA methylation was investigated as a potential player in the variation in response described both within an individual over time as well as across individuals. Fibroblast exposure to 5-aza-2'-deoxycytidine, a DNA demethylating agent, increased the cellular response to LPS, but more so in cultures that had previously displayed low responding phenotypes. This suggested that DNA methylation acted as an inhibitor of the innate immune response, and may be responsible for some degree of the variation seen in the LPS response. To determine the effect of epigenetic factors on this response, microarray analysis was conducted on RNA isolated from cells either having been epigenetically modified (DNA demethylation and histone hyperacetylation) or without undergoing any epigenetic treatment. This analysis identified 1,758 genes with altered expression due to epigenetic modification. To focus on DNA methylation's role, methylated CpG island recovery assay (MIRA-Seq) libraries were created from fibroblasts to investigate differential methylation from a group of the same individuals sampled at 5 and 16 months of age. In addition, transcriptomic data were generated by RNA-Seq from fibroblasts collected from the young and older samples and exposed to LPS for 0, 2, and 8 hours to characterize age-associated changes in the innate immune response. Cultures from older animals were much more responsive to LPS as indicated by greater expression of IL-8, IL-6, TNF-α, and CCL20 at various times in response to LPS. TLR4 and CD14 were more highly expressed in older cultures, suggesting these fibroblasts are more able to detect the presence of LPS. Analysis of the bovine fibroblast methylome revealed methylation with remarkable stability except for 20 regions along the genome undergoing major shifts due to age. Similar data were collected from fibroblasts isolated from different individuals displaying either a low or high responding phenotype resulting in 843 regions with differential methylation between groups. This suggests that DNA methylation may be playing a role in both the age-dependent and between animal differential responses to LPS, and also gives the first in depth look at the bovine fibroblast methylome and its stability over time.

Asthma is a multi-faceted chronic inflammatory disease accompanied by loss of airway epithelial integrity leading to remodeling of the airways. Perturbations to the lung redox environment, including alterations in glutathione (GSH) content, have been reported in asthma. GSH can be conjugated to protein cysteines, controlling protein function in an oxidant-dependent process known as protein S-glutathionylation (PSSG). The thioltransferase, glutaredoxin-1 (Glrx1), deglutathionylates proteins under physiological conditions, restoring sulfhydryl groups of target proteins. Glrx1 is emerging as a critical player in settings of allergic airway disease, but its function in regulating epithelial cell responses to asthma-relevant cytokines has not been examined. Furthermore, the role of Glrx1 in controlling the extent of airway remodeling in response to house dust mite (HDM) in vivo is still not well understood. Interleukin-17A (IL-17A) is a potent cytokine that stimulates epithelial cells to produce pro-inflammatory mediators, in part by activating the nuclear factor kappaB (NF-κB) pathway, a key regulator of inflammation. We demonstrate that interleukin-17A (IL-17A) induces rapid activation of both classical and alternative NF-κB, while simultaneously resulting in protein oxidation and PSSG. In particular, we show IL 17A induces S-glutathionylation of RelA (RelA-SSG) and IKKα (IKKα-SSG), which is enhanced following siRNA-mediated knockdown of Glrx1. We also demonstrate that absence of Glrx1 leads to increased nuclear content of RelA and RelB and enhanced production of NF-κB-driven pro-inflammatory genes, KC and CCL20 while decreasing IL-6 expression. Finally, we show that siRNA-mediated knockdown of IKKα attenuates nuclear RelA and RelB and dampens pro-inflammatory gene production. Together, these data indicate a crucial role for the Glrx1/PSSG axis in controlling RelA-SSG, IKKα-SSG and epithelial cell responsiveness to IL-17A. Mice lacking Glrx1 were previously shown to display enhanced resolution of allergic airway disease induced by ovalbumin (Ova) challenge. In this study, we determined the role of Glrx1 in a HDM model of allergic airway disease. Wild type (WT) mice and Glrx1 deficient (Glrx1-/-) mice demonstrated similar total lung cell counts, but Glrx1-/- mice displayed fewer neutrophils than WT mice. Conversely, mice overexpressing Glrx1 specifically in CCSP positive cells in the lung (Epi-Glrx1) showed attenuated total lung cell counts and lung eosinophils compared to control mice. Immunohistological analysis of remodeling markers revealed that Glrx1-/- mice displayed increased HDM-induced mucus metaplasia, α smooth muscle actin (αSMA) positivity and collagen staining compared to WT mice. Evaluation of total lung collagen showed that Glrx1-/- mice had significantly higher collagen content compared to WT mice. In Epi-Glrx1 mice, attenuation of mucus metaplasia, αSMA content and collagen staining was observed compared to control mice. Furthermore, Epi-Glrx1 mice also demonstrated significantly impaired collagen production compared to control mice. We also demonstrate that Glrx1 absence results in decreased expression of the epithelial cell marker, E-cadherin, and increased expression of αSMA, a mesenchymal marker. Together, these studies demonstrate a critical role for Glrx1 in controlling epithelial cell responses to IL-17A and in mediating in vivo collagen production in response to chronic allergen exposure.

Doctor of Philosophy / Department of Industrial & Manufacturing Systems Engineering / Chih-Hang Wu / The topic of infectious disease epidemics has recently attracted substantial attentions in research communities and it has been shown that the changes of human behaviors have significant impacts on the dynamics of disease transmission. However, the study and understanding of human reactions into spread of infectious disease are still in the very beginning phase and how human behaviors change during the spread of infectious disease has not been systematically investigated. Moreover, the study of human behaviors includes not only various enforced measures by public authorities such as school closure, quarantine, vaccination, etc, but also the spontaneous self-protective actions which are triggered by risk perception and fear of diseases. Hence, the goal of this research is to study the impacts of human behaviors to the epidemic from these two perspectives: spontaneous behavioral changes and public intervention strategies. For the sake of studying spontaneous changes of human behaviors, this research first time applied evolutionary spatial game into the study of human reactions to the spread of infectious disease. This method integrated contact structures and epidemics information into the individuals’ decision processes, by adding two different types of information into the payoff functions: the local information and global information. The new method would not only advance the field of game theory, but also the field of epidemiology. In addition, this method was also applied to a classic compartmental dynamic system which is a widely used model for studying the disease transmission. With extensive numerical studies, the results first proved the consistency of two models for the sake of validating the effectiveness of the spatial evolutionary game. Then the impacts of changes of human behaviors to the dynamics of disease transmission and how information impacts human behaviors were discussed temporally and spatially. In addition to the spontaneous behavioral changes, the corresponding intervention strategies by policy-makers played the key role in process of mitigating the spread of infectious disease. For the purpose of minimizing the total lost, including the social costs and number of infected individuals, the intervention strategies should be optimized. Sensitivity analysis, stability analysis, bifurcation analysis, and optimal control methods are possible tools to understand the effects of different combination of intervention strategies or even find an appropriate policy to mitigate the disease transmission. One zoonotic disease, named Zoonotic Visceral Leishmaniasis (ZVL), was studied by adopting different methods and assumptions. Particularly, a special case, backward bifurcation, was discussed for the transmission of ZVL. Last but not least, the methodology and modeling framework used in this dissertation can be expanded to other disease situations and intervention applications, and have a broad impact to the research area related to mathematical modeling, epidemiology, decision-making processes, and industrial engineering. The further studies can combine the changes of human behaviors and intervention strategies by policy-makers so as to seek an optimal information dissemination to minimize the social costs and the number of infected individuals. If successful, this research should aid policy-makers by improving communication between them and the public, by directing educational efforts, and by predicting public response to infectious diseases and new risk management strategies (regulations, vaccination, quarantine, etc.).

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly-pathogenic mouse-passaged amebae (Mp) and weakly-pathogenic axenically-grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium from Mp and Ax amebae were the presence of an activity band of approximately 58kDa and 100 kDa bands susceptible to the action of cysteine and metallopeptidase inhibitors, respectively. Further roles of the peptidases during the invasion process were examined by in vitro invasion assays in the presence of inhibitors and Cysteine and metallopeptidase inhibitors were found to greatly reduce invasion through the ECM. This study establishes a functional linkage of the expressed peptidases to the invasion process, and these peptidases may serve as a candidate target for therapeutic management of N. fowleri infection.

Dexamethasone has been shown to inhibit IgE-mediated mast cell activation, and the present research investigated its role in suppressing IL-33-mediated mast cell activation. We have found that micromolar concentrations of Dexamethasone are capable of suppressing IL-33-mediated mast cell cytokine production, on several genetic backgrounds, and in not only bone marrow derived mast cells, but also peritoneal mast cells. Intracellular staining demonstrated that Dexamethasone significantly reduces expression of the IL-33 receptor, T1/ST2, in mast cells; however, the cytokine suppression is independent of T1/ST2 downregulation. At the same time, Dexamethasone pretreatment significantly reduced ERK phosphorylation, but our data suggests that inhibition occurs even prior to ERK blockade. Finally, Dexamethasone treatment in vivo reduced IL-33-mediated cytokine production and neutrophil infiltration in the murine peritoneum. Thus, Dexamethasone, a well-established therapy for inflammatory disease, can suppress IL-33-mediated mast cell activation, and may therefore be effective for treating diseases now being attributed to IL-33 effects.

Kainate receptor knockout mice have unique differences within their immune system. They exhibit an attenuated TH2 branch, while maintaining a robust TH1 response. Specifically, blocking the formation of functional kainate receptors affects mast cells and their related pathologies. While they seem to develop and activate normally in vivo and in vitro, KAR KO mast cells release more inflammatory mediators upon degranulation. These mice experience severe anaphylactic shock due to two compounding abnormalities. First, KAR KO mast cells release significantly more histamine in vivo upon IgE-mediated activation. Second, the animals over-respond to exogenous histamine with drastic temperature drops compared to WT. This report shows that the kainate receptor plays an important role in mast cell-mediated immune responses.