<p>Originally considered the sole providence of protein coding sequences, evolutionary biology has begun to recognize the importance of non-coding DNA in dictating phenotypic adaptation. Exclusively examined in eukaryotic anatomical development, <em>cis</em>-regulatory modifications have the power to alter the spatial-temporal dynamics of gene expression without the plieotropic consequences of protein modification. Owing to the need to integrate horizontally acquired DNA into existing regulatory networks, <em>cis</em>-regulatory mutations may also significantly contribute to prokaryotic evolution. The horizontal acquisition of <em>Salmonella</em> Pathogenicity Island (SPI)-2 led to the evolutionary divergence of <em>Salmonella enterica</em> from <em>S. bongori</em>. Use of the type 3 secretion system encoded in SPI-2 allowed <em>S. enterica</em> to exploit an intracellular host niche offered by immune cells and allowed for its systemic dissemination. Here we identify ancestrally encoded <em>srfN</em> and <em>dalS</em> and demonstrate that through acquisition of a binding site for the SPI-2 regulator, SsrB, they have contributed to the pathoadaptation of <em>S. enterica</em> to the host environment. We also demonstrate that ancestral regulatory networks contribute to the establishment of an expression hierarchy for SPI-2 <em>in vitro</em> and to transcriptional priming in the host lumen prior to invasion. These findings demonstrate that <em>cis</em>-regulatory modifications have significantly contributed to the evolution of <em>S. enterica</em> as an intracellular pathogen.</p> / Doctor of Philosophy (PhD)

<p>Dendrimers provide an ideal scaffold for molecular imaging and therapeutics due to their mono-disperse structure and easily modifiable core, interior, and periphery. The controlled-stepwise synthesis leads to perfect, defined architectures that can easily be modified to incorporate targeting and imaging moieties. Specifically poly (2,2-bis(hydroxymethyl)-propanoic acid) (PMPA) dendrimer structures exhibit excellent aqueous solubility, low toxicity, biocompatibility and biodegradability, which are necessary requirements for an ideal <em>in vivo</em> imaging scaffold.</p> <p>Fever of unknown origin (FUO) is a common condition involving elevated temperatures above 101°F, which goes undiagnosed after a week of investigation. The primary causes of FUO are infection and cancer, however methods of diagnosis are non-specific and quite slow. Developing a method to detect bacterial infections, and therefore rule out more severe conditions such as cancer, would be very useful in diagnosis of this condition. Approximately two thirds of infection cases in hospitals are determined to be caused by one of six pathogens known as the ESKAPE pathogens and developing a molecular imaging probe that would detect these specific pathogens would be a very useful FUO diagnostic tool.</p> <p>Siderophores are naturally occurring molecules that exhibit a high affinity for Fe³⁺, and are effective at entering bacterial cells after complexing iron. In particular, Desferal, a commercially available siderophore used for iron chelation therapy, has been successfully modified, radiolabeled, and studied as an imaging agent. Dendrimers were modified with Desferal and used to investigate the effect of multivalent display of siderophores on a single macromolecular structure.^1-3</p> <p>We herein discuss the preparation of a series of siderophore-terminated PMPA dendrimers that were radiolabeled and studied to compare bacterial uptake between a monomeric siderophore and a macromolecule displaying multiple siderophores on its periphery. To introduce Desferal to the periphery of a dendrimer, activated p-nitrophenyl carbonates were used. A series of Desferal-terminated dendrimers of generations 1-3 was synthesized in yields of 59-89 % and evaluated for suitability as an infection-imaging probe.</p> <p>The Desferal-terminated dendrimer series was evaluated for its affinity to iron(III) and gallium(III), as well as tested for steric hindrance effects at the periphery. The series was successfully radiolabeled with ⁶⁷Ga using mild conditions and <em>in vitro</em> bacterial uptake studies were performed with <em>Staphylococcus aureus</em>, one of the ESKAPE pathogens, to determine if multivalency increases bacterial uptake.</p> <p>Preliminary results indicate that the poor water solubility of the Desferal-terminated dendrimer series needs to be improved in order to increase bacterial uptake of the compounds, however viable candidates for metal chelation were successfully produced.</p> / Master of Science (MSc)

Background: Clostridium difficile is the most common cause of healthcare-associated infections in the United States, and has been associated with adverse outcomes in the acute care setting. However, little is known regarding the burden or impact of C. difficile infection (CDI) in long-term acute care hospitals (LTACHs). Methods: A retrospective matched cohort study was performed among patients at an urban, university-affiliated LTACH between July 2008 and October 2015. The incidence rate of LTACH-onset CDI was assessed and patient characteristics associated with adverse outcomes examined. Patients with CDI were matched to concurrently hospitalized LTACH patients without a diagnosis of CDI. A multivariable model using logistic regression was developed to determine characteristics associated with a composite primary outcome of either 30-day readmission to an acute care hospital or mortality. Subgroup analyses were performed for patients with a diagnosis of severe CDI. Results: The overall incidence of CDI was 21.4 cases per 10,000 patient-days. Patients with CDI had a mean age (±SD) of 70 ±14 years and a mean admission Charlson Comorbidity Index (CCI) of 4 ±2. Median (IQR) time between admission and diagnosis of CDI was 16 days (range: 9-23 days). In the final multivariable model, CDI was not a significant risk factor for the primary outcome (OR, 1.06 [95% confidence interval {CI}, 0.53-2.10]). Congestive heart failure (OR, 2.27 [95% CI, 1.15-4.57]), albumin level (OR, 0.44 [95% CI, 0.22-0.79]), and immunosuppression (OR, 2.94 [95% CI, 1.06-8.39]) were independent risk factors for the primary outcome. On subgroup analysis, severe CDI and CCI were significant risk factors for the primary outcome in bivariable analysis (OR, 2.91 [95% CI 1.03-8.20] and OR, 1.36 [95% CI 1.06-1.80], respectively). Only CCI remained significant in the multivariable model (OR, 1.32 [95% CI 1.02-1.75]). Conclusions: LTACH-onset CDI was found to have a relatively high incidence in an urban, university affiliated LTACH. CDI was not a significant risk factor for the composite outcome of 30-day readmission or mortality. Future research should focus on infection prevention and antibiotic stewardship measures to decrease CDI specifically in the LTACH setting. / Epidemiology

Systemic lupus erythematosus (SLE) is complex autoimmune disease in which autoantibodies form against double stranded DNA (dsDNA) and nuclear antigens. Autoantigen immune complexes form, deposit in the vasculature, and cause multisystem organ damage. Both genetic and environmental factors contribute to the development of SLE. This thesis will explore three major themes found in the study of SLE: 1) Bacterial infection as an environmental trigger, 2) cytokine dysregulation in immune cells, and 3) the treatment of end organ damage in the form of lupus nephritis. Viral infections have long been associated with the development of systemic autoimmune disease, but the mechanisms by which chronic bacterial infections may promote autoimmunity remain unclear. In chapter three we show that a component of bacterial biofilms, the amyloid-like protein “curli”, irreversibly forms fibers with bacterial or eukaryotic DNA during biofilm formation. This interaction accelerates amyloid polymerization and creates potent immunogenic complexes that activate immune cells, including dendritic cells, to produce cytokines such as type I interferons, which are pathogenic in SLE. When given systemically, curli/DNA composites trigger immune activation and production of autoantibodies in lupus-prone and wild type mice. We also found that infection with curli-producing bacteria triggered higher autoantibody titers in lupus-prone mice compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity. Cytokine dysregulation is also common in SLE patients. Serum cytokines are often elevated during active disease, including type I IFNs and IL-10. In chapter four we demonstrate that Il10 is a type I IFN response gene and has increased basal expression in dendritic cells (DCs) derived from pre-disease lupus-prone Sle1,2,3 mice. We show that Sle1,2,3-derived DCs overproduce IL-10 in response to TLR ligands and that this is the result of autocrine signaling though the type I IFN receptor (IFNAR). These results suggest that dysregulation of cytokine signaling in the myeloid compartment may contribute to IL-10 dysregulation in SLE. Renal disease remains a major cause of morbidity and mortality in SLE. A number of mouse models of chronic kidney disease have implicated the EGFR-family receptors in the progression of renal fibrosis and dysfunction. In chapter five we show that renal expression of ErbB2 is increased in murine lupus. We therefore asked if EGFR-family inhibition could prevent murine lupus nephritis. To test this possibility we used lapatinib, an EGFR-ErbB2 dual kinase inhibitor, in an IFN-accelerated model of murine lupus. We found that lapatinib administration lowered autoantibody levels but worsened renal disease. Lapatinib failure to treat murine lupus nephritis despite lowered autoantibody levels suggests EGFR-family signaling is required for tissue repair in the acute phase of kidney injury. Together this thesis clearly demonstrates the complexity of systemic autoimmune disease – bringing us to the crossroads of immunity and tolerance. The combination of both environmental triggers (e.g. bacterial infection) and genetic susceptibility (e.g. intrinsic cytokine dysregulation) leads to end organ damage (e.g. lupus nephritis). Here we sought to explore each aspect of disease progression in the hopes to develop better interventions for systemic autoimmune disease. / Microbiology and Immunology

No / Despite wide acknowledgment that hand hygiene is the pre-eminent measure in the control of health-care-associated infection, Didier Pittet and colleagues 1 have highlighted that there is still a need for a systematic programme of research that will allow the development of new¿as well as refinement of existing¿approaches to hand cleansing. One of the key priorities Pittet and colleagues identified is the need for investigations into the relative importance of between and within patient cross-transmission

Toxoplasma gondii is a protozoan parasite that causes life-threatening disease in congenitally infected infants and immunocompromised patients, such as those inflicted with AIDS. Toxoplasmic encephalitis (TE) is a common presenting condition in an AIDS infection. People become infected with T. gondii by ingesting tissue cysts in undercooked meats or by ingesting oocysts excreted by cats. Methylmercury (MeHg) is a well-documented neurotoxicant that accumulates in the brain and causes severe mental and visual dysfunction, including chronic encephalopathy. Consumption of contaminated fish, grains, and seeds are common sources of human exposure to methylmercury. Studies from our laboratory suggest that oral exposure to a single high dose of 20 mg/kg MeHg does not increase the susceptibility to acute toxoplasmosis in CBA/J mice. Therefore, we further investigated endpoints associated with immunotoxicity and neurotoxicity in 6-week old, female CBA/J mice exposed to both MeHg and T. gondii during a chronic T. gondii infection. We examined both single and multiple doses of MeHg exposure in a chronic parasitic infection model. In the single high dose study, four groups of six-week-old, female CBA/J mice were either fed 25 T. gondii tissue cysts of the ME-49 strain or given vehicle. Six weeks later, two out of the four groups (T. gondii and vehicle control) were orally gavaged with a single dose of 20 mg/kg body weight of MeHg and sacrificed seven days post exposure. Experiments from the multiple MeHg dose study were performed under similar conditions with the same number of groups and dosed by oral gavage with 8 mg/kg body weight of MeHg on days 0, 2,4,7,10,13. These mice were sacrificed on day 17 or 18 after initiating MeHg exposure. Flow cytometry following exposure to a single dose of MeHg in mice with a chronic T. gondii infection revealed significant changes (P < 0.05) within the T cell subpopulation percentages caused by exposure to MeHg. For example, the thymic CD4+CD8+ T cell subpopulations were increased (P <0.05). However, MeHg had no significant effect on the CD4+CD8-, CD4-CD8+, or non-T cell subpopulations in the spleen. Furthermore, MeHg increased splenic cellularity and spleen-to-body-weight ratios with or without a concurrent T. gondii infection. MeHg also caused a significant decrease in mouse body weight. There was a significant (P <0.05) increase in brain tissue cyst counts within the group exposed to both MeHg and T. gondii (16 ± 4, mean ± SE, n=7) versus T. gondii alone (4 ± 1, n=8). Histopathological examination demonstrated that the brain was affected, as lesions, gliosis, and meningitis were notable in mice given T. gondii. Exposure of mice to multiple doses of MeHg also resulted in effects on the immune system of CBA/J mice with and without chronic toxoplasmosis. Total cellularity and numbers of CD4+CD8+, CD4+CD8-, CD4-CD8+, and CD4-CD8- T-cell subpopulations show a marked decrease in number in the thymus, while total cellularity was also decreased in the spleen following concurrent exposure to T. gondii and MeHg. Flow cytometric examination of lymphocyte populations (CD4+ and CD8+ lymphocytes) in the spleen and thymus demonstrated differences from control in the groups exposed to T. gondii and MeHg. Histopathological examination did not reveal any significant lesions. The data from experiments in which single or multiple doses of MeHg were given to mice with a chronic T. gondii infection indicate that concurrent exposure, to both MeHg and T. gondii, dependent on dose and time of exposure had notable effects, especially on the immune system (Supported by NIH Grant F36GM20301). / Ph. D.

Southwest Virginia has recently become an emerging focus of activity for La Crosse (LAC) virus, a mosquito-transmitted arbovirus in the California serogroup of Bunyaviruses. In 2005 and 2006, ovitrap surveys were conducted to access the spatiotemporal oviposition activity of LAC virus vectors Aedes triseriatus, Ae. albopictus and Ae. japonicus across a wide region of southwest Virginia. Egg abundance and oviposition patterns of these vectors were significantly different across the three study areas. The primary LAC virus vector, Ae. triseriatus, was collected in the greatest abundance from all three areas, and favored forested habitats. Aedes albopictus was the second most abundant species collected, and was found to favor urban environments. Aedes japonicus also has a preference for urban habitats, and is actively expanding its range throughout southwest Virginia. Dogs were used to determine their efficacy as sentinels for assessing the distribution of LAC virus in southwest Virginia. Canine serum samples were tested using plaque reduction neutralization (PRNT) assays. Of the 436 collected canine serum samples, 21 (4.8%) were positive for LAC virus antibodies. LAC virus seroprevalence was evident in dogs from each study region, including areas where LAC virus human cases and LAC virus positive mosquito isolates have not been reported. As a result, this study provided documentation of horizontal transmission of LAC virus throughout southwest Virginia, demonstrating that dogs make useful sentinels for assessing the distribution of LAC virus in an area. The final objective examined the effects of coinfection with D. immitis microfilariae and LAC virus in three species of Aedes mosquitoes. No significant differences were found between mosquitoes fed dually infected bloodmeals (i.e. D. immitis microfilariae and LAC virus) and those fed bloodmeals containing LAC virus only. A follow-up study found low mosquito midgut penetration rates by D. immitis, despite using biologically significant doses of microfilariae. Failure to demonstrate enhancement of LAC virus in vector mosquitoes suggests that D. immitis does not have a significant impact on LAC virus epidemiology in areas where these organisms co-exist. / Ph. D.

Microfilarial enhancement of viral transmission is well documented, however only one previously studied model used components that occur together in nature and therefore has realistic implications. La Crosse (LAC) virus encephalitis is the most common mosquito-borne illness affecting children in the United States. LAC virus is prevalent in the Great Lake and Mid-Atlantic states and coincidently this area overlaps the region of highest infection for Dirofilaria immitis, the nematode that cause canine heartworm disease. Ae. albopictus and Oc. triseriatus are important vectors of La Crosse virus and among the numerous species able to transmit D. immitis. In this study, Aedes albopictus and Ochlerotatus triseriatus were infected with La Crosse virus and Dirofilaria immitis to determine the effects of dual infection on the dissemination and transmission of the virus. The effects of dual infection varied between the species tested. Ae. albopictus had significantly higher tolerance to D. immitis infection than Oc. triseriatus. Dissemination for dually infected Ae. albopictus were higher than the control group for all days tested, except one. Transmission rates for D. immitis infected Ae. albopictus were significantly higher than the control group on day 14 post infection. No microfilarial enhancement of viral dissemination or transmission was observed for Oc. triseriatus. The infection, dissemination, and tranmission rates were low for both species compared to rates of previous studies. Low rates could be a result of low susceptibility for the strains tested. In a second study, mosquitoes were collected from two counties in Southwestern Virginia to determine the abundance of potential La Crosse virus and D. immitis vector species. The abundance and distribution of mosquito species were examined in 2003 and 2004 using gravid traps. An unexpected finding was the significant increase in the abundance of Ochlerotatus japonicus. In 2003, collections were made over 192 trap nights from June to August yielding 5,879 mosquitoes of which only 24 were Oc. japonicus. In 2004, 12,151 mosquitoes were trapped from June to September over 160 trap nights. Oc. japonicus was the second most abundant mosquito species and the dominant Ochlerotatus species collected in gravid traps. Oc. japonicus was collected in low numbers in June, but the abundance increased significantly in July and remained consistent throughout the rest of the season. Of the other major mosquito species collected in this study, only Aedes albopictus exhibited a similar seasonal pattern as Oc. japonicus. Other biological similarities of Oc. japonicus and Ae. albopictus are discussed. / Master of Science in Life Sciences

Dengue viral infection is a mosquito-borne disease with four distinct strains, where the interactions between these strains have implications on the severity of the disease outcomes. The two competing hypotheses for the increased severity during secondary infections are antibody dependent enhancement and original antigenic sin. Antibody dependent enhancement suggests that long-lived antibodies from primary infection remain during secondary infection but do not neutralize the virus. Original antigenic sin proposes that T cells specific to primary infection dominate cellular immune responses during secondary infections, but are inefficient at clearing cells infected with non-specific strains. To analyze these hypotheses, we developed within-host mathematical models. In previous work, we predicted a decreased non-neutralizing antibody effect during secondary infection. Since this effect accounts for decreased viral clearance and the virus is in quasi-equilibrium with infected cells, we could be accounting for reduced cell killing and the original antigenic sin hypothesis. To further understand these interactions, we develop a model of T cell responses to primary and secondary dengue virus infections that considers the effect of T cell cross-reactivity in disease enhancement. We fit the models to published patient data and show that the overall infected cell killing is similar in dengue heterologous infections, resulting in dengue fever and dengue hemorrhagic fever. The contribution to overall killing, however, is dominated by non-specific T cell responses during the majority of secondary dengue hemorrhagic fever cases. By contrast, more than half of secondary dengue fever cases have predominant strain-specific T cell responses. These results support the hypothesis that cross-reactive T cell responses occur mainly during severe disease cases of heterologous dengue virus infections. Finally, using the results from our within-host models, we develop a multiscale model of dengue viral infection which couples the within-host virus dynamics to the population level dynamics through a system of partial differential equations. We analytically determine the relationship between the model parameters and the characteristics of the solutions, and find thresholds under which infections persist in the population. Furthermore, we develop and implement a full numerical scheme for our model. / Ph. D.

Yes / Dog bite injuries are a common cause of patient presentation to NHS emergency departments (EDs) and minor injuries units, and are generally associated with a low level of acuity, despite an inherent capacity for significant soft tissue damage to be inflicted by canine jaws capable of exerting terrific bite forces. Anatomical sites for injury correlate to victim age, with hand and wrist injuries predominating in the adult population. The most common complication is infection secondary to inoculation of oral flora, with the hands being particularly vulnerable due to their anatomy. Injuries to structures such as tendons can be discreet, and retained foreign bodies can easily be overlooked. Wound care has a propensity to attract a disproportionately high level of malpractice actions, and approaches to the management of dog bite injuries have largely been empirical, which may render the practitioner particularly exposed. In response to increasing pressures on healthcare systems, paramedics with extended scopes of practice, including wound care and suturing, are being utilised to assess, manage, treat, and either refer or discharge patients with apparently minor injuries, in strategies aimed at reducing hospital admissions. This article adopts a case study format to examine and evaluate treatment modalities and the current evidence base informing best practice in terms of dog bite injuries from the perspective of a paramedic practitioner, with critical reflection on the decision making process and complexities of such episodes of care in the pre-hospital setting.