Yersinia pestis, the gram-negative causative agent of plague, is a master of immune evasion. The bacterium possesses a type three secretion system which translocates Yop effector proteins into host immune cells to inhibit a number of immune and cell signaling cascades. Interestingly, this apparatus is not expressed at low temperatures such as those found within the flea vector and is therefore neither in place nor functional when the bacteria are first transmitted into a mammalian host. However, the bacterium is still able to avoid activating the immune system, even very early during infection. When grown at 37°C (human body temperature) Y. pestis produces a tetra-acyl lipid A molecule, which is antagonistic to the human Toll like receptor 4/MD2, the major lipopolysaccharide recognition receptor. Although tetra-acyl lipid A binds this receptor complex, it does not induce signaling, and in fact inhibits the receptors interaction with other stimulatory forms of lipid A. The work undertaken in this thesis seeks to determine if the production of tetra-acyl lipid A by Y. pestis is a key virulence determinant and was a critical factor in the evolution of Y. pestis from its ancestral parent Yersinia pseudotuberculosis. By examining the enzymes involved in the lipid A biosynthesis pathway, it has been determined that Y. pestis lacks LpxL, a key enzyme that adds a secondary acyl chain on to the tetra acyl lipid A molecule. In the absence of this enzyme, Y. pestis cannot produce a TLR4 stimulating form of lipid A, whereas Y. pseudotuberculosis does contain the gene for LpxL and produces a stimulatory hexa acyl lipid A. To determine if the absence of LpxL in Y. pestis is important for virulence, LpxL from E. coli and Y. pseudotuberculosis were introduced into Y. pestis. In both cases the addition of LpxL led to bacterium which produced a hexa-acylated lipid A molecule and TLR4/MD2 stimulatory LPS. To verify the LpxL phenotype, lpxL was deleted from Y. pseudotuberculosis, resulting in bacteria which produce tetra-acylated lipid A and nonstimulatory LPS. Mice challenged with LpxL expressing Y. pestis were found to be completely resistant to infection. This profound attenuation in virulence is TLR4 dependent, as mice deficient for this receptor rapidly succumb to disease. These altered strains of the bacterium also act as vaccines, as mice infected with Y. pestis expressing LpxL then challenged with wild type Y. pestis do not become ill. These data demonstrate that the production of tetra-acyl lipid A is a critical virulence determinant for Y. pestis, and that the loss of LpxL formed a major step in the evolution of Y. pestis from Y. pseudotuberculosis. These bacterial strains were also used as tools to determine the contributions of different innate immune receptors and adaptor molecules to the host response during Y. pestis infection. The use of LpxL expressing Y. pestis allowed identification of the innate immune pathways critical for protection during Y. pestis infection. This model also established that CD14 recognition of rough LPS is critical for protection from Y. pestisexpressing LpxL, and activation of the IL-1 receptor and the induction of IL-1β plays a major role in this infection as well. The lipid A acylation profile of gram negative bacteria can have a direct and profound effect on the pathogenesis of the organism. This work illustrates a previously unknown and critical aspect of Y. pestis pathogenesis, which can be extended to other gram-negative pathogens. The greater detail of the contributions which different host adaptor and receptor molecules make to the overall innate immune signaling pathway will allow a better insight into how gram negative infections progress and how they are counteracted by the immune system. Alterations of the lipid A profile of Y. pestis have important implications for the production of vaccines to Y. pestis and other gram negative pathogens. Taken together, this work describes a novel mechanism for immune evasion by gram negative bacteria with consequences for understanding the immune response and the creation of more effective vaccines, both of which will decrease the danger posed by this virulent pathogen.

Vaccinia virus (VAC) is the prototypical member of the orthopoxvirus genus of the poxvirus family and the virus used for smallpox vaccinations. The following describes the testing of VAC variants designed to have similar immuno-protective profiles with decreased pathogenicity, examines the immune response to VAC after lethal infection in wild type and lupus-prone mice, and describes a method that allows for the enumeration of VAC-specific CD8+ T in naïve and VAC-immune mice. The first part describes work examining VAC Wyeth (VAC-Wy) variants engineered to be less pathogenic in vivo. VAC-Wy variants included genes that code for three immunomodulatory proteins, an interferon-γ (IFNγ) binding protein (B8R), an interleukin 18 (IL-18) binding protein (C12L), and a complement binding protein (C3L, or C21L) or various combinations of the three knockouts, and a triple knockout (VAC-Wy -/-/-) in which all three genes were knocked out of a variant virus. The immunomodulatory effects of other IFNγ binding proteins on VAC-Wy pathogenesis in the mouse were also examined. Virus recombinants where the B8R gene was replaced with a truncated mouse IFNγ receptor gene or a gene that encodes a B8R/IFNγ fusion that allows for dimerization of the secreted IFNγ receptor were studied. As the knockouts and variants were made in the current vaccine VAC-Wy strain, only high dose (1x107 PFU’s) intra nasal (I.N.) infection of mice reliably resulted in detectable virus in the lungs. Further testing revealed that all knockout and variant viruses grew to similar levels after high dose I.N. infections. Protection induced by vaccination with the VAC-Wy variants was studied in comparison to immunizations with the VAC-Wy parental strain. Mice were immunized by tail skin scarification to mimic human immunizations, and this was followed months later by I.N. challenge with 20 LD50’s of VAC-WR. All VAC-Wy recombinants tested, including the VAC-Wy -/-/-, provided similar levels of protection as the parental VAC-Wy strain from a lethal VAC-WR I.N. infection. Mice immunized with the VAC-Wy -/-/- induced similar amounts of neutralizing antibody and similar numbers of CD8+ T cells specific to a subdominant determinant as VAC-Wy. While examining high dose, normally lethal, VAC-WR I.N. infections, a profound splenic CD8+ T cell immune suppression was noted that might have been caused by Fas dependent activation induced cell death (AICD). Using high dose intra-peritoneal (I.P.) and I.N. models of VAC-WR infection, decreased weight loss, decreased virus titers, and increased T cell numbers were found in Fas mutant (B6.MRL-Faslpr/J) mice in comparison to B6 wild type mice on day 6. It would be expected that Fas-deficient CD8+ T cells from B6.MRL-Faslpr/J mice (B6-lpr) would survive a high dose VAC-WR infection better than CD8+ T cells that could express Fas if T cells were being eliminated by Fas-dependent AICD, but co-adoptive transfer experiments using splenocytes from B6-lpr and B6.Cg- IgHaThy-1aGPi-1a/J (IgHa) wild type counterparts found no difference in the numbers or proliferation of donor CD8+ T cells at day 6. As the B6-lpr mice were better protected from VAC-induced weight loss early after lethal VAC-WR infections, it was possible that B6-lpr mice might be protected early in infection. In fact, Fas mutant mice had decreased virus loads in the fat pads, livers, and spleens in comparison to B6 wild type mice at days 2 and 3. In addition to the decreased virus titers, the severe splenic lymphocyte deficiency noted in B6 wild type mice as early as day 2 after high dose I.P. infection was ameliorated in B6-lpr mice. Further experiments demonstrated that uninfected B6-lpr mice had increased numbers of memory phenotype (CD44+) CD4+, CD8+ and γδ+ T cells, with an increased number of γδ+ T cells and NK cells in splenic lymphocytes in comparison to wild type B6 mice. Uninfected B6-lpr mice also had increased numbers of IFNγ+ CD8+ T cells after polyclonal stimulation with an antibody against CD3ε. In lymphocyte depletion experiments performed at day 3, antibody depletion of CD4, CD8, or NK or treatment with an antibody that was specific to the γδ+ TCR did not significantly alter virus loads in B6-lpr mice. In co-adoptive transfer experiments, splenocytes from wild type or B6-lpr mice survived high dose VAC-WR challenge similarly suggesting that B6- lpr splenocytes were not intrinsically better protected from lymphocyte depletion by lack of the Fas protein. On day 2 after high dose I.P. VAC-WR infection, B6- lpr mice had increased numbers of IFNγ+ NK cells, IFNγ+ CD8+ T cells, and IFNγ+ CD4+ T cells. B6-lpr and B6 mice treated with an antibody against IFNγ had significantly increased virus titers in the spleens and livers. Interestingly, there was no significant difference in liver or spleen virus titers when comparing anti- IFNγ antibody treated B6 mice or anti-IFNγ antibody treated B6-lpr mice. These results suggest that multiple leukocyte populations co-operatively or redundantly provide B6-lpr mice with increased protection from high dose VAC-WR infections through increased production of IFNγ. The third part of this work describes the enumeration of total numbers of pathogen-specific CD8+ T cells in a mouse through use of an in vivo limiting dilution assay (LDA). The extensive proliferation of virus-specific CD8+ T cells that occurs after virus infection was used to enumerate numbers of virus-specific CD8+ T cells in a naïve mouse. By transferring limiting amounts of carboxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1+Ly5.2+ heterogeneous CD8+ T cells into Thy1.2+Ly5.1+ hosts, CD8+ T cell precursor frequencies to whole viruses can be calculated. The calculations are based on finding the number of donor CD8+ T cells that results in CFSElo (i.e. proliferated) donor CD8 T cells in 50% of the hosts. Using probit or Reed and Muench 50% endpoint calculations, CD8+ T cell precursor determinations were made for naïve and immune states to a virus challenge. It was found that in naïve B6 mice, 1 in 1444 CD8+ T cells proliferated in response to VAC-WR (~13,852 VAC-WR-specific CD8+ T cells per mouse) and 1 in 2956 proliferated in response to lymphocytic choriomeningitis virus (LCMV) (~6,761 LCMV-specific CD8+ T cells per mouse). In mice immune to VAC-WR, the number of VAC-WR-specific LDA precursors, not surprisingly, dramatically increased to 1 in 13 (~1,538,462 VAC-WR- specific CD8+ T cells per mouse) consistent with estimates of VAC-WR-specific memory T cells. In contrast, precursor numbers to LCMV did not increase in VAC-WR-immune mice (1 in 4562, ~4384 LCMV-specific CD8+ T cells in a VAC-WR-immune mouse) consistent with the fact that VAC-WR provides no heterologous immunity to LCMV. Using H-2Db-restricted LCMV GP33-specific P14 transgenic T cells it was found that, after accounting for take of donor T cells, approximately every T cell transferred underwent a full proliferative expansion in response to an LCMV infection and a high efficiency was also seen in memory populations. This suggests that most antigen-specific T cells will proliferate in response to infections at limiting dilution. These results, which are discussed in comparison to other methods, show that naïve and memory CD8+ T cell precursor frequencies to whole viruses can be remarkably high. In total this work further advances knowledge of the immunity, pathogenesis, and prevention of poxvirus infections. This was accomplished by studying VAC-Wy recombinants as improved vaccines, by examining the mechanisms and cell types important in early protection from high dose poxvirus infections in B6 and B6-lpr mice, and by describing a method to enumerate total numbers of virus-specific CD8+ T cells in a mouse.

Health impact assessment (HIA) has emerged in the U.S. as one promising process to increase social and environmental justice through addressing health equity issues within planning. HIA practice is guided by values such as democracy and equity and grounded in broad social determinants of health. The most readily applied definition of democracy is problematic because it implies an element of direct, participatory engagement with the public. This is at odds with HIA practice that largely relies on stakeholder engagement strategies. This dissertation critically examines the engagement strategies of three transportation planning HIA cases to more fully understand how the HIA process may or may not promote democratic values and protect community health interests. It employs a multi-case study design that uses qualitative content analysis to trace community health interests through the HIA process, HIA document, and target plan. It finds that while the field is overstating the participatory nature of HIA, commitments to health equity and broad determinants of health protect community health interests with and without robust engagement of community stakeholders.

Humans experience ubiquitous exposures to estrogenic environmental chemicals from food, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. In our study, pregnant CD-1 mice were orally exposed to BPS or ethinyl estradiol (EE2, a positive control for estrogenicity) from gestational day 9 through postnatal day (PND) 2, the period when many estrogen-sensitive organs are developing. After weaning, the offspring were administered either oil (vehicle) or an estrogen challenge (1 μg EE2/kg/day) for ten days starting at PND21 (prior to puberty), PND80 (early adulthood), or PND260 (later adulthood). Timing of puberty was evaluated in females by noting the date on which vaginal opening occurred. After the 10 day estrogen challenge, we evaluated the response of endocrine sensitive organs through measurements of organ weight, tissue morphology, and gene expression in both males and females. We observed dose- and sex-specific effects of BPS and EE2 treatment, as well as alterations in the responses of males and females to the estrogen challenge. This study sheds light on the effects of low dose xenoestrogen exposures on estrogen-sensitive organs including the reproductive tract and mammary gland. Furthermore, it improves our understanding of the influence of environmental chemicals on secular trends of earlier age of puberty in girls reported over the past few decades.

Tick populations have been immigrating into northeast Tennessee from east Tennessee, Kentucky, Virginia, and North Carolina. Counties in states bordering northeast Tennessee harbor tick species associated with human illness. Human diseases transmitted by ticks include ehrlichiosis, spotted fever rickettsial group diseases, tularemia, anaplasmosis, babesiosis, Lyme disease, alpha-gal syndrome, Heartland virus, Powassan virus, and southern tick-associated rash illness (STARI). These diseases cause morbidity and mortality in human populations and may pose a high risk to individuals, wildlife, and livestock. The Cherokee National Forest covering the east Tennessee border provides a permissible environment for ticks to immigrate and thrive. Residents of northeast Tennessee frequently use the natural environment for a variety of purposes, creating exposure risk at the human-animal-environment interface. This study performed a scoping review and meta-analysis addressing topics informing epidemiological investigation of tick populations. The meta-analysis identified geography, climate, and Shannon-Wiener Diversity Index as the most significant variables associated with northeast Tennessee tick populations. Additionally, tick surveillance in northeast Tennessee counties was performed. These counties included Carter, Greene, Hancock, Hawkins, Johnson, Washington, and Unicoi. Primary tick species present in the summer included the American Dog tick (Dermacentor variabilis); the winter included the Blacklegged/Deer tick (Ixodes scapularis). Canonical correlation analysis was used to identify which environmental variables had the most influence, to what degree, and in a positive or negative direction. Altitude, total forest land, forest canopy, and fraction of surface water area were statistically significant. More altitude was correlated with more clinical cases; less total forest land, canopy, and fraction of surface water area was correlated with less clinical cases. Lastly, species distribution modeling of the invasive Asian longhorned tick was conducted. Study results indicate a low to moderate risk for tick-borne illness exposures among human populations, which is poised to increase. Species distribution modeling and clinical case data reports suggested an increasing exposure risk from improved habitat suitability. Increased risk is related to climate change and tick population growth in metropolitan areas. Finally, surveillance and control methods are summarized for integration into public health interventions.

<p>Diarrhoea due to the consumption of unsafe drinking water is a major cause of death worldwide, despite many small and large-scale water, sanitation and hygiene (WASH) intervention programs and policy processes. Many Sub-Saharan African communities have relied on WASH interventions by governmental and non-governmental organizations to reduce the burden of diarrhoeal diseases, however they often fail to be sustainable.</p> <p>Safe drinking water is achieved by protecting/treating water at all points along the drinking water supply chain (DWSC), from the source to the point-of-use. Gathering data on the sanitary environment and microbiological quality of water along the DWSC can support the design of water quality interventions. In addition, an examination of the knowledge, attitudes and practices (KAP) of local people on WASH topics could support the design of more socioculturally relevant interventions. The purpose of this research was to develop and pilot a simple yet economical and robust method to inform more socioculturally relevant water quality interventions in rural Sub-Saharan Africa, and to test whether variation in the risks existed at the neighbourhood-scale within three neighbourhoods of a single community in rural Kenya.</p> <p>The results of this study demonstrated that practices, which affect water quality in the DWSC, varied at the neighbourhood-scale. For example, source water quality was poor in the three study neighbourhoods, however the hazards and contaminating practices that posed a risk to water quality varied (i.e., bathing, toileting, laundry). Household water quality was also poor and at risk in all three neighbourhoods, however the practices that represented a risk to household water quality varied (e.g. storage conditions, sanitation practices). Female water collectors were knowledgeable on the causes of diarrhoea, however their preferred approaches toward WASH intervention approaches varied by neighbourhood. The collection and analysis of neighbourhood-scale social and environmental WASH data is therefore recommended for the prioritization and design of appropriate interventions to improve water quality.</p> / Master of Applied Science (MASc)

Background: Sperm contain highly compact nuclei, inhibiting DNA extraction using traditional techniques. Current methods extracting sperm DNA involve lengthy lysis and no means of stabilizing DNA, hindering clinical research. Objective: We sought to optimize an efficient method of extracting high quality human sperm DNA. Methods: Sperm from three volunteers were isolated using PureCeption. We tested 1) proteinase K with DNA/RNA Shield, 2) DTT and TCEP as reducing agents, 3) QIAshredder homogenization, and 4) stability of sperm DNA fresh (baseline) or after 4 weeks of storage at 4OC in DNA/RNA Shield using modified Quick-gDNA MiniPrep. DNA was PCR amplified using ALU primers and digested with Hinf1 restriction enzymes. DNA methylation was measured by MassARRAY. Results: DNA concentrations were similar with (30.1+0.28ng/μL, 33.4+0.21ng/μL) and without (28.9+0.00ng/μL, 30.9+0.85ng/μL) proteinase K. Sperm cells were lysed after 1 and 20 minutes with 25mM TCEP and 100mM DTT respectively. TCEP (50mM) produced greater DNA concentrations (17.2+0.50ng/μL, 21.3+0.71ng/μL) than 50mM DTT (12.6+0.28ng/μL, 12.3+0.35ng/μL). Adding QIAshredder to 50mM TCEP increased DNA concentrations (25.9+0.35ng/μL, 21.7+0.49ng/μL versus 18.6+0.99ng/μL, 12.3+0.35ng/μL). At baseline and 4 weeks: 1) DNA concentrations were similar (36.2+2.75 ng/μL, 32.2+1.38ng/μL, 44.3+3.93ng/μL versus 40.0+2.98ng/μL, 37.6+1.38ng/μL, 38.7+3.93ng/μL respectively) and 2) DNA was equally amplified by PCR and digested with restriction enzymes. DNA methylation was similar at baseline and 4 weeks for SNURF (1.43+1.02%, 1.55+0.95%), PEG10 (3.69+0.66%, 4.28+1.52%), and H19 (88.93+3.24%, 91.78+2.00%). Conclusions: We stabilized and isolated high quality DNA from human sperm using 5 minute versus > 2 hour lysis in other methods. Our methods may facilitate efficient clinical research.

Nurses are frequently seen in public in their “scrubs,” which could mean that contaminated clothing is being brought into the community, thereby posing an infection risk. The purpose of this study is to investigate if and which contaminants are present on the fabrics and the actions nurses are taking to eliminate contamination risks. Eleven scrub tops were worn on hospital units over one twelve-hour shift. The contaminated scrubs and three control tops were then swabbed and used to inoculate agar plates. After incubation, colonies were counted, streaked onto nutrient and Mannitol-salt agar for isolation, and incubated. Using API Staph strips and Gram staining, the bacteria were identified. The nurses also completed a short survey on laundering and scrub care. All scrub tops, except the controls, were contaminated with multiple species of bacteria including Staphylococcus species. Responses to the survey showed that no two nurses washed their scrubs in the same manner and many wear them in public. The results determined that bacteria can survive on clothing and pose the possibility of transmission throughout the hospital and public venues. The survey results indicate a need for employer laundering policies, public awareness of the risk for transmission of disease from contaminated clothing, and stricter regulations about employees wearing scrubs outside of health care facilities.

Exposure to mercury poses significant risks to the health of humans and wildlife. Globally, coal-fired power plant (CFPP) is a major source of mercury emissions, with China being the largest contributor to global atmospheric mercury. As a signatory country of the Minamata Convention on Mercury, China is developing its National Implementation Plan on Mercury Control, which gives priority to control of mercury emissions from CFPPs. While social benefits play an important role in designing environmental policies in China, the potential public health and economic benefits of mercury control in the nation are not yet understood, mainly due to the scientific challenges to trace mercury’s emissions-to-impacts path. Moreover, little is known about the potential benefits for the neighboring countries in East Asia resulted from China’s mercury control. This study evaluates the health and economic benefits for China and neighboring countries in East Asia from mercury reductions from China’s CFPPs. Four representative mercury control policy scenarios are analyzed, and the evaluation is explicitly conducted following the policies-to-impacts path under each policy scenario. We link a global atmospheric model to health impact assessment and economic valuation models to estimate economic gains for China and its three neighboring countries (Japan, South Korea and North Korea) from avoided mercury-related adverse health outcomes under the four emission control scenarios, and also take into account the key uncertainties in the policies-to-impacts path. Under the most stringent control scenario, the cumulative benefit of the mercury reduction by 2030 is projected to be $430 billion for the four countries together (the 95% confidence interval is $102-903 billion, in 2010 USD). Our findings suggest that although China is the biggest beneficiary of the mercury reduction in CFPPs, neighboring countries including Japan, South Korea and North Korea can also benefit (~7% of the total benefits) from China’s mercury reduction.

Background: Exposure to perfluorooctane sulfonic acid (PFOS) or to perfluorooctanoic acid (PFOA) increases mouse and human peroxisome proliferator–activated receptor alpha (PPARα) subtype activity, which influences lipid metabolism. Because cholesterol is the substrate from which testosterone is synthesized, exposure to these substances has the potential to alter testosterone concentrations. Objectives: We explored associations of total testosterone and sex hormone–binding globulin (SHBG) concentrations at age 15 years with prenatal exposures to PFOS, PFOA, perfluorohexane sulfonic acid (PFHxS), and perfluoronanoic acid (PFNA) in females. Methods: Prenatal concentrations of the perfluoroalkyl acids (PFAAs) were measured in serum collected from pregnant mothers at enrollment (1991–1992) in the Avon Longitudinal Study of Parents and Children (ALSPAC). The median gestational age when the maternal blood sample was obtained was 16 weeks (interquartile range, 11–28 weeks). Total testosterone and SHBG concentrations were measured in serum obtained from their daughters at 15 years of age. Associations between prenatal PFAAs concentrations and reproductive outcomes were estimated using linear regression models (n = 72). Results: Adjusted total testosterone concentrations were on average 0.18-nmol/L (95% CI: 0.01, 0.35) higher in daughters with prenatal PFOS in the upper concentration tertile compared with daughters with prenatal PFOS in the lower tertile. Adjusted total testosterone concentrations were also higher in daughters with prenatal concentrations of PFOA (β = 0.24; 95% CI: 0.05, 0.43) and PFHxS (β = 0.18; 95% CI: 0.00, 0.35) in the upper tertile compared with daughters with concentrations in the lower tertile. We did not find evidence of associations between PFNA and total testosterone or between any of the PFAAs and SHBG. Conclusions: Our findings were based on a small study sample and should be interpreted with caution. However, they suggest that prenatal exposure to some PFAAs may alter testosterone concentrations in females.