Browsing by Author "Ryan, Elizabeth, committee member"
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Item Open Access A case study of a wheat-free diet on autoimmune disease progression(Colorado State University. Libraries, 2014) Connor, Trevor, author; Cordain, Loren, advisor; Hickey, Matthew, committee member; Ryan, Elizabeth, committee member; Schenkel, Alan, committee memberBackground and Aims: Autoimmune disease encompasses a broad range of over 80 conditions for which only three have an identified environmental trigger. Gliadin is the trigger in celiac disease, a condition that has been linked to other autoimmune conditions including Crohn's disease and type I diabetes (T1D). The purpose of this study was to investigate case studies of autoimmune patients who employed a wheat-free or Paleo-style diet (WFP) to manage their conditions. Methods: A descriptive case study was performed that utilized questionnaires administered online and medical records from autoimmune disease patients who had consumed a WFP diet. Results: Fifty-seven patients were evaluated in the study (mean age 37.3 yrs., SD 10.1), including 23 males and 34 females, 24 of whom provided medical records. Thirty of the 57 volunteers demonstrated signs of disease improvement while consuming a WFP. The rates varied across conditions with eight of eight Crohn's disease patients experiencing remission, while three of four T1D patients exhibited signs of improvement. Five of 15 patients with ankylosing spondylitis, rheumatoid arthritis, or undifferentiated and multiple connective tissue disorders worsened while on the diet. Conclusions: Patients with single organ autoimmune diseases previously linked to increased intestinal permeability showed the greatest improvement after consuming a WFP. The results of these case studies warrant further controlled research examining the effects of wheat consumption on Crohn's disease and T1D.Item Open Access Anatomic plasticity and functional impacts of neural – immune and neural – epithelial signaling in the intestine(Colorado State University. Libraries, 2021) Schwerdtfeger, Luke A., author; Tobet, Stuart A., advisor; Chicco, Adam, committee member; Myers, Brent, committee member; Ryan, Elizabeth, committee memberThe intestinal wall is a multicompartmental barrier tissue composed of over 25 distinct cell types with integrated and complex signaling both within and between compartments. The gut wall is also a large endocrine organ comprised of cells capable of producing dozens of peptides used for hormonal and other signaling functions. However, the mechanistic roles that neural secretions play in regulating the gut epithelial barrier in health and disease are not well known. Additionally, frequently used models available for studying intestinal function outside of the body lack the complexity to investigate neural – epithelial and neural – immune signaling interactions. Using a bifurcated approach to method development, we created two culture systems for maintaining the full thickness of the intestinal wall ex vivo. One method allows for culture of mouse or human organotypic intestinal slices that maintain the gut wall for 6 or 4 days, respectively. This system does not however, maintain a true luminal – epithelial barrier as seen in the in vivo gut. The second method, a microfluidic organotypic device (MOD) enables maintenance of explanted mouse or pig intestinal tissue for up to 3 days ex vivo, with an intestinal barrier intact. These two methods allow for investigating and cross-validating of numerous biological questions now previously possible using traditional culture models. Neuronal fiber proximity to gut epithelia has been shown, with goblet, tuft and enteroendocrine cells being closely opposed by fibers. Goblet cells secrete mucopolysaccharides, a first line of defense separating luminal microbiota from host tissue. I have recently shown that vasoactive intestinal peptide (VIP) can regulate goblet cell production in organotypic slices of mouse ileum. This peptide is also in close proximity to Paneth cells in the base of the crypt, and enteric mast cells. There were sex differences in baseline mast cell neuronal proximity, quantities, and cell size in mouse ileum. Further, mast cells showed a sex difference in responses to lipopolysaccharide challenge. Further investigation of neurosecretory factor regulation of immune and epithelial function is needed, both in goblet cells and other secretory epithelia like anti-microbial producing Paneth cells, and in immune components like mast cells. Graphical illustration of the dissertation project is included below.Item Open Access Combining curcumin and alpha-lipoic acid to treat cardiometabolic syndrome(Colorado State University. Libraries, 2015) Binns, Scott Edward, author; Bell, Christopher, advisor; Ryan, Elizabeth, committee member; Hickey, Matthew, committee memberINTRODUCTION: Obesity is associated with increased risk of developing cardiometabolic disease characterized by decreased insulin sensitivity, positive energy balance, and cardiovascular disease. Separately, the dietary compounds curcumin (CUR) and alpha lipoic acid (ALA) increase energy expenditure and insulin sensitivity. However the efficacy of combined CUR and ALA supplementation to improve characteristics of cardiometabolic health in overweight humans has never been evaluated. METHODS: 11 overweight and sedentary adults were randomly assigned to a placebo (n=5; age: 44 ± 8 years; body mass index: 31.9 ± 2.1 kg/m²; VO₂peak: 26.8 ± 4.1 ml/kg/min; (mean ± SE)) or CUR+ALA (n=6; age: 38 ± 7 years; body mass index: 32.2 ± 1.2 kg/m²; VO₂peak: 24.9 ± 3.7 ml/kg/min) 12-week intervention. Measurements of resting metabolic rate (RMR) and thermic effect of feeding (TEF) via indirect calorimetry, and insulin sensitivity via the hyperinsulinemic euglycemic clamp technique were assessed in a pre/post treatment manner. Additionally, resting blood pressure, and body composition via dual energy x-ray absorptiometry (DEXA) were measured. RESULTS: 12-weeks of CUR+ALA supplementation did not change RMR (1467 ± 89.9 to 1539 ± 94.4 kcal/day; P=0.239) or TEF AUC (P=0.51). Additionally, no change in glucose infusion rate occurred as a result of supplementation (12.8 ± 2.1 to 12.4 ± 1.2 mg/FFM kg/min; P=0.690). Resting systolic blood pressure was unaffected in the CUR+ALA group (116 ± 3.2 to 123.8 ± 25.4 mmHg; P=0.31). Body fat percentage did not change with CUR+ALA supplementation (43.8 ± 2.2 to 41.6 ± 1.9 body fat percent; P=0.162), however, body fat percentage decreased in all subjects regardless of treatment (P=0.002). DISCUSSION: These preliminary data do not support the use of CUR+ALA to improve metabolic and cardiovascular health in overweight adults.Item Open Access Development and application of an improved in vitro model for aerosol toxicology(Colorado State University. Libraries, 2014) Hawley, Brie, author; Volckens, John, advisor; Dow, Steven, committee member; Ryan, Elizabeth, committee member; Reynolds, Stephen, committee memberIn vitro cellular studies offer an economical and rapid screening tool for assessing aerosol toxicity. Traditional submerged in vitro cell models and exposure techniques are often criticized for their inability to (1) simulate in vivo cellular morphology (2) maintain the chemical and physical characteristics of sampled aerosol and (3) estimate 'delivered' exposure levels. Further, the exposure levels applied in traditional submerged in vitro systems are often orders of magnitude above inhalational exposures that occur in vivo. Improved airway cell culture models and direct air-to-cell exposure systems have been developed over the last few decades; these improvements offer greater 'real-world' significance to in vitro aerosol toxicology. Air-liquid interfaced airway cell cultures offer greater physiological relevance than previous, submerged cell cultures. Further, direct air-to-cell exposure systems offer the ability to (1) better maintain the chemical and physical characteristics of test aerosols and (2) more closely control and approximate exposure levels. Presented here, are two improved direct air-to-cell aerosol exposure systems that rely upon electrostatic deposition or gravitational settling to directly expose well-differentiated airway cell cultures to three different aerosols of interest, with regard to occupational and environmental health. The first and second study presented here used electrostatic deposition to expose well-differentiated normal human bronchial epithelial cells to diesel particulate matter and complete diesel exhaust. Cells were exposed to either (1) diesel particulate matter or (2) complete diesel exhaust from an engine run on either petro- or biodiesel, and with and without a diesel particulate filter. Cellular response was assessed by measuring transcripts associated with inflammation, oxidative stress, aromatic hydrocarbon response and overall cellular dysfunction at 1, 3, 6, 9, and 24 hours after exposure to diesel particulate matter. Cellular response to complete diesel exhaust was assessed by measuring transcripts associated with oxidative stress and aromatic hydrocarbon response at two hours after exposure. The main aims of these two studies were to (1) characterize the time course of the proinflammatory response of normal human bronchial epithelial cells after exposure to diesel particulate matter and (2) screen for the effects of exposure to petro- and biodiesel exhaust, with and without a diesel particulate filter. The third study presented here used gravitational settling to expose well-differentiated human bronchial or nasal epithelial cells to two different particle size fractions from inhalable dust collected at a local dairy parlor. Cellular response was assessed by measuring transcripts associated with inflammation at two hours after exposure. Cell compromise was also measured in all three studies by measuring percent lactate dehydrogenase release. Significant airway cellular responses were observed in all three studies, at levels of exposure far lower than reported in previous traditional in vitro studies. Results from the work presented here strongly support the use of improved airway cell models and direct air-to-cell exposure systems in future in vitro studies in aerosol toxicology.Item Open Access Development of Lactobacillus acidophilus as an oral vaccine vector and effects of rice bran ingestion on the mucosal health of Malian infants(Colorado State University. Libraries, 2020) Vilander, Allison C., author; Dean, Gregg, advisor; Abdo, Zaid, committee member; Dow, Steven, committee member; MacNeill, Amy, committee member; Ryan, Elizabeth, committee memberMost pathogens enter the body at the mucosa and induce innate and adaptive immune responses at these surfaces essential for protection against infection and disease. Induction of mucosal immune responses is best achieved locally but mucosal vaccines have been difficult to develop with few currently approved for use. Almost all are attenuated live vaccines which limits their use and efficacy in some populations. Strategies to enhance the mucosal immune response to vaccination and move away from attenuated live vaccines are needed. Prebiotics (nondigestible food ingredients that promotes growth of beneficial microorganisms) and probiotics (live microorganisms that are beneficial when ingested) are an active area of interest for improving mucosal health and increasing oral vaccine performance. Here we present the development of the probiotic Gram-positive lactic acid bacteria Lactobacillus acidophilus (LA) as a novel oral subunit vaccine. LA has many advantages as an oral vaccine vector including endogenous acid and bile resistance, heat tolerance, and numerous proteins that interact with the mucosal immune system. We show that LA can induce immune responses to weakly immunogenic neutralizing peptides from HIV-1 and rotavirus. To enhance the immune response, we developed the E. coli type I pilus protein, FimH, as a LA vaccine adjuvant. FimH increased the immune response to vaccination and increased LA trafficking by antigen presenting cells to the mesenteric lymph node, an important site of mucosal immune induction. We also evaluate the effects of ingestion of the nutrient dense prebiotic rice bran on mucosal health in a cohort of healthy Malian infants at risk for malnutrition and the subclinical condition environmental enteric dysfunction. Rice bran ingestion was found to decrease episodes of diarrhea, decrease the age to elevated fecal microbiome α-diversity, and stabilize total fecal secretory IgA concentrations over time. These results indicate that rice bran protects from diarrhea and improves the mucosal environment.Item Open Access Examination of the complex relationships among dietary components, type II diabetes, weight change, and breast cancer risk among Singaporean Chinese women(Colorado State University. Libraries, 2015) Canales, Lorena Lea, author; Peel, Jennifer, advisor; Clark, Maggie, committee member; Bachand, Annette, committee member; Nelson, Tracy, committee member; Ryan, Elizabeth, committee memberType II diabetes and breast cancer are on the rise in Asian populations that have typically had lower burdens of disease. Intake of dietary components high in nutrients with anti-oxidative and anti-inflammatory properties, such as green tea, soy, fruits and vegetables, may protect against the development of type II diabetes and may improve HbA1c (glycated hemoglobin) levels, a clinically relevant biomarker of diabetes and prediabetes. Furthermore, modifiable lifestyle factors such as diabetes, weight change and diet that influence endogenous hormone levels and the insulin pathway may play a role in the development of breast cancer. This dissertation includes three aims that examined different aspects of the complex relationships between diet, diabetes, weight change, and breast cancer risk in the Singapore Chinese Health Study, a prospective cohort study that enrolled 63,257 Chinese men and women aged 45-74 years between 1993 and 1998. First, we examined the association between intake of green tea, soy, and a vegetable-fruit-soy dietary pattern on HbA1c levels among self-reported, nondiabetic men and women, examined separately (Aim 1). We also evaluated type II diabetes and weight change (separately) in relation to risk of breast cancer, as well as the potential interaction of diet (soy and green tea intake) with the exposures of interest among women only (Aims 2 and 3). Dietary intake was assessed at baseline (1993-1998) by in-person interviews using a validated 165-item food frequency questionnaire. HbA1c levels were measured from blood samples collected in the follow-up period after baseline enrollment (1999-2004), and self-reported diabetes diagnosis was determined at the follow-up interview. Self-reported weights at the baseline and follow-up interviews were used to determine weight change. Multivariable linear regression (Aim 1) and proportional hazards regression models (Aims 2 and 3) were used to evaluate these associations. In Aim 1, adjusted mean HbA1c levels were inversely related to soy protein intake (p-value = 0.02; p for trend across the four quartiles of soy protein intake = 0.05) among women; the mean HbA1c difference between the highest and lowest quartile of soy protein intake of 0.07%. We also observed higher HbA1c levels for women with higher green tea intake (p for trend of 0.11), which was in the direction opposite to that hypothesized. In Aim 2, we observed a non-statistically significant increase in breast cancer risk among women with type II diabetes (adjusted hazard ratio [HR]=1.24, 95% confidence interval [CI]: 0.82, 1.86). The assessment of the joint effects of diabetes and lower soy isoflavone intake suggested a weak non-significant interaction between these variables on breast cancer risk; the HR for breast cancer was slightly elevated among those with lower soy isoflavone intake, while among those with higher isoflavone intake the HR was consistent with a null association. There was no evidence of interaction when evaluating soy food, soy protein and green tea intake on the diabetes and breast cancer association. In Aim 3, we did not observe evidence of an increase in breast cancer risk among women reporting weight gain between baseline and follow-up interviews; however, we observed an increase in risk among women who lost between 3 and 5 kilograms between baseline and follow-up interviews (HR=1.31, 95% CI: 0.94, 1.83), which was in the direction opposite of what was hypothesized. This result was similar when we removed breast cancer cases diagnosed within the first two years of follow-up. There was no evidence of interaction between weight change and soy and green tea intake. In conclusion, we provide suggestive evidence that soy protein intake is associated with decreased HbA1c levels among self-reported nondiabetic women. Furthermore, our results suggest that soy isoflavone intake may weakly modify the association between type II diabetes and breast cancer risk. Collectively, the results of these three studies indicate that soy intake may be protective for the development and progression of type II diabetes and could also attenuate the adverse impact of type II diabetes on breast cancer risk. However, given that these results are suggestive for different soy components and the short follow-up time of the prospective evaluation of breast cancer risk, further research is needed to investigate this question. Furthermore, research among populations with varying levels of soy intake is also needed to assess these associations.Item Open Access HIV and Zika: modeling pathogenesis and therapies in humanized mice(Colorado State University. Libraries, 2017) Charlins, Paige, author; Akkina, Ramesh, advisor; Liber, Howard, committee member; Aboellail, Tawfik, committee member; Ryan, Elizabeth, committee memberInvestigation into pathogenesis of critical viral diseases that pose a global threat is important not only for pathogen modeling but also for treatment discovery. Use of humanized mice to study viral pathogens, including HIV-1 and Zika virus, provide a novel insight into the mechanisms of viral persistence and act as a platform for therapeutic intervention and observation. In the context of HIV-1 research, humanized mice are ideal for parroting the infection mechanics. Recapitulation of HIV-1 viral pathogenesis enables the study of various facets of the virus lifecycle, concerning infection establishment, detection, and treatment. In this respect, humanized mouse modeling of HIV-1 infection is paramount for continued studies of HIV-1 in terms of HIV-1 latency, with focus on viral reactivation and outgrowth, and for research with novel therapeutic approaches for treatment and modulation of the infection. The model has been paramount in development of an ultra-sensitive assay for detection of latent HIV-1 and validation of aptamer-siRNA conjugates and conditionally replicating vectors for treatment of HIV-1 infection. Humanized mice can further be expanded for the study of additional emerging pathogens, such as Zika virus, in terms of infection pathology and immune response. Infection of humanized mice with a Puerto Rican strain of Zika virus resulted in high plasma viral loads detectable up to four months post inoculation with wide spread dissemination detected in various tissues by IHC and CLARITY. Understanding these different elements of various viral infections with the ability to establish innovative perspective into mechanics and therapeutics, contribute a previously inaccessible view of human specific viral pathogenesis and treatment.Item Open Access Investigation of surface interactions of pyrazinamide and pyrazinoic acid with synthetic and natural lipid membrane model systems(Colorado State University. Libraries, 2022) Gasparovic, Nathaniel, author; Crans, Debbie C., advisor; Henry, Chuck, committee member; Ryan, Elizabeth, committee memberPyrazinamide (PZA) is a pro-drug used in the treatment of tuberculosis. Upon administration of the drug, it is converted to its active form of pyrazinoic acid (POA) by the tuberculosis bacterium; this is believed to be the biologically active form of the drug which exerts anti-tubercular activity. However, it is generally accepted that both compounds interact with and transverse the membrane, with POA potentially functioning as a protonophore and lowering the intracellular mycobacterial pH. To investigate the interactions of PZA and POA in model membranes, we employed Langmuir monolayers to investigate the potential membrane-disrupting effects of PZA and POA on a model membrane. At physiological pH, neither PZA nor POA disrupted the membrane, although a difference in compressibility was observed. At acidic pH, POA became more disruptive but only at high, non-physiological concentrations. 1H NMR spectroscopy of a microemulsion system was used to investigate the location of PZA and POA in the interface in different protonation states. The neutral POA species was found to preferentially reside in the interface while the charged species remained in the interfacial water. Finally, the effects of PZA and charged POA on the bilayer in liposomes were investigated. A leakage assay on fluorophore-filled liposomes showed that PZA and POA do not induce leakage in the membrane at physiological conditions.Item Open Access Investigations of radiation-induced and spontaneous chromosomal inversion formation and characteristics(Colorado State University. Libraries, 2014) Cartwright, Ian Michael, author; Kato, Takamitsu, advisor; Bailey, Susan, committee member; Bedford, Joel, committee member; Ryan, Elizabeth, committee member; Tu, Anthony, committee memberTo view the abstract, please see the full text of the document.Item Open Access Metabolomic profiles of Oryza sativa and influence of genetic diversity(Colorado State University. Libraries, 2011) Heuberger, Adam Lawrence, author; Brick, Mark, advisor; Leach, Jan, committee member; Ryan, Elizabeth, committee member; Byrne, Patrick, committee member; Thompson, Henry, committee memberFood crops with enhanced health characteristics are being developed in many breeding programs. Rice (Oryza sativa L.) is an ideal candidate to study traits related to health due to its importance as both a global staple food and a model system for cereal crops. Evaluating metabolite profiles can be a high-throughput method to identify variation in health properties of dietary components. Metabolomics is a useful tool to assess the influence of genetics on total metabolite variation in the cooked grain. Cooked rice metabolite profiles for 10 diverse varieties were determined using ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS) on aqueous-methanol extracts. A total of 3,097 molecular features were detected, and 25% of the features varied among the 10 varieties (ANOVA, p < 0.001). Both z-score and partial least squares-discriminant analysis (PLS-DA) showed variation consistent with subspecies-based varietal groupings, and indicated genetic control over the metabolite profiles. Variation in total phenolics and vitamin E was also consistent with varietal groupings. Genes in biochemical pathways for health-related metabolites were interrogated for allelic variation by single nucleotide polymorphisms (SNPs). SNP variation may serve as an important mechanism by which genes influence metabolic variation. The influence of genetic diversity on the metabolite profile of the rice grain was also assessed for two interacting effects: genotype-environment interactions (GEI) and genotype-fermentation interactions (GFI). GEI was assessed by growing two diverse rice varieties in the field and the greenhouse. Gas-chromatography-MS (GC-MS) was used to detect primary metabolites from aqueous-methanol extracts of cooked rice. Genotype, environmental, and GEI effects were observed for many metabolites, including the amino acid phenylalanine, a precursor for many secondary metabolites related to human health. Genes associated with phenylalanine synthesis were screened in rice gene expression databases, and variation within and among the genes suggests they are a potential source of genetic variation for phenylalanine synthesis. Both the metabolite and gene expression patterns indicate a potential interaction between phenylalanine and serine synthesis. The GC-MS data implies the GEI effects on primary metabolism may correspond to variation in secondary metabolites that are predicted to affect human health. Additionally, human health attributes of the grain may be dependent on fermentation of rice metabolites by gut microorganisms. GFI effects were assessed by fermenting three highly similar rice varieties with Saccharomyces boulardii, a probiotic yeast. Metabolites were extracted and detected by GC-MS. A PLS-DA model showed evidence of fermentation (F) effects, but not GFI. However, when extracts were assessed for the ability to inhibit viability of lymphoma cells, both F and GFI effects were apparent. It is therefore likely that GFI effects may exist among diverse rice varieties, and that interactions affect the bioactivity of rice metabolites. In summary, total metabolite variation is largely influenced by the rice genotype, including interactions with environment and fermentation. These data describe both heritable and non-heritable sources of variation. Thus, although genetic variation in rice is sufficient to establish metabolite profiles specific to human health characteristics, the heritability of a secondary metabolite-associated health trait is likely influenced by both environment and fermentation effects.Item Open Access Microbes in the mucosa: impacts of the mucosal immune system and oral vaccination with Lactobacillus acidophilus on the gut microbiome(Colorado State University. Libraries, 2021) Fox, Bridget E., author; Dean, Gregg, advisor; Abdo, Zaid, advisor; Tobet, Stuart, committee member; Ryan, Elizabeth, committee memberThe mucosal immune system is constantly balancing between the clearance of pathogens, tolerance of self-antigen and food, and maintenance of homeostasis within the microbiota. Vaccination via mucosal routes is advantageous because it provides protection at local mucosal sites and systemically. However, induction of efficacious responses are often difficult due to the inherent barriers of the mucosal tissues. We have developed a probiotic-based mucosal vaccination platform that utilizes recombinant Lactobacillus acidophilus (rLA) to overcome these obstacles presented in oral vaccination. Here, we sought to determine whether repeated administration of rLA alters the intestinal microbiome as a result of L. acidophilus probiotic activity (direct competition and selective exclusion) or from the host's mucosal immune response against the rLA vaccine. To address the latter, IgA-seq was employed to characterize shifts in IgA-bound bacterial populations. Additionally, we determined whether using rice bran as a prebiotic would influence the immunogenicity of the vaccine and/or IgA bound bacterial populations. Our results show that the prebiotic influenced the kinetics of rLA antibody induction, and that the rLA platform does not cause lasting disturbances to the microbiome. Nucleotide-binding oligomerization domain containing 2 (NOD2) has presented itself as an essential regulator of immune responses within the gastrointestinal tract. This innate immune receptor is expressed by several cell types, including both hematopoietic and nonhematopoietic cells within the gastrointestinal tract. Mice harboring knockouts of NOD2 only in CD11c+ cells were used to better characterize NOD2 signaling during mucosal vaccination with rLA. We show that NOD2 signaling in CD11c+ cells is critical for mounting a humoral immune response against rLA. Additionally, disruption of NOD2 signaling in CD11c+ cells results in an altered bacterial microbiome profile in both vaccinated and unvaccinated mice.Item Open Access Modeling the evolution of SIV into HIV using humanized mice(Colorado State University. Libraries, 2016) Boddeda, Srinivasa Rao, author; Akkina, Ramesh, advisor; Laybourn, Paul, committee member; Juarrero, Mercedes Gonzalez, committee member; Ryan, Elizabeth, committee memberAcquired Immunodeficiency syndrome (AIDS) is caused by two lentiviruses belonging to the family Retroviridae, namely HIV-1 and HIV-2, which originated from SIVcpz and SIVsmm respectively. Multiple independent cross-species transmission events of SIV from chimpanzees (SIVcpzPtt ) and gorillas (SIVgor) have given rise to four groups of HIV-1 (M, N, O, P), while transmission from sooty mangabeys (SIVsmm) is responsible for at least 8 groups of HIV-2 (A-H) (Ayouba et al., 2013; Gao et al., 1999; Hirsch et al., 1989). Some of these groups are extremely rare. However, four have established themselves in human populations as pandemic (HIV-1 group M) or epidemic (HIV-1 group O, HIV-2 groups A and B) outbreaks (Faria et al., 2014). While these data suggest that SIV transmission to a human host is by itself not sufficient to establish a new epidemic outbreak, it also implies that viral adaption is necessary for efficient spread of the virus within humans (Marx et al., 2004). However, exact role of factors such as immune selective pressure and genomic changes, contributed to successful SIV adaptation in humans remains unclear. Study of SIV transmission to humans has been limited by lack of an appropriate model. Ethical constraints prevent experimental challenge of human subjects with SIV and transmission studies have thus far been limited to non-human primates (NHPs) (Chahroudi et al., 2012). While NHP studies have been instrumental to identify key differences in innate and adaptive immune responses that influence transmissibility and virulence of SIV between species, until now experimental in vivo challenge of a functional human immune system with SIV has not been possible. Recently however, a new generation of humanized Rag2−/−γc−/− mice has been established that supports systemic engraftment of a functional human immune system (Akkina, 2013; Denton et al., 2012). These animals are susceptible to HIV-1 infection by mucosal routes and display key features of pathogenic HIV-1 infection in humans, including sustained plasma viremia, CD4+ T cell depletion and increased levels of immune exhaustion markers (Berges et al., 2006; Palmer et al., 2013). Additionally, humanized mice are able to mount adaptive immune responses to HIV-1 infection, producing antibodies and HIV-1 specific T cell responses. In the current study, we used humanized Rag2−/−γc−/− mice to study SIV transmission to humans. We hypothesized that humanized mice could support SIV infection, and that the presence of in vivo selective pressures, either innate or adaptive, would drive viral evolution. That is, we expected the majority consensus virus emerging in the plasma of productively infected humanized mice different from the stock virus used to infect the animals. Viral adaptation within infected humanized mice could produce a variant virus more fit for growth and transmission in humans. We infected humanized mice with both SIVsmmE041 and SIVcpzLB715 isolates and measured plasma viral loads and determined complete viral genome sequences. Both SIVsmm and SIVcpz showed stable viral loads over time for up to 7 months. SIVsmm virus from the infected mice was successfully passaged to three successive generations of humanized Rag2−/−γc−/− mice via intraperitoneal route. We were also able to show that human cell-adapted SIVsmm can infect humanized mice through the mucosal route. Sequence analyses of SIVsmm and SIVcpz output virus from these mice showed many functional mutations in various genes such as Gag, Pol, Env, Vif, Vpx, Vpr and Nef regions. In both viruses, Env regions showed the highest number of mutations suggesting that the envelope region of this virus might be under selective pressure for this virus to be able to replicate in human cells. We observed that the number of mutations in all genes increased over time, suggesting that the SIVsmm virus is continuously evolving and adapting for successful replication and transmission in these humanized mice. These studies will provide a flexible in vivo model for elucidating the mechanisms underlying SIV transmission and gain of function to replicate in humans.Item Open Access Murine models of Staphylococcus aureus biofilm infections and therapeutic protein A vaccination(Colorado State University. Libraries, 2013) Walton, Kelly D., author; Kendall, Lon V., advisor; Dow, Steve W., advisor; Ryan, Elizabeth, committee memberStaphylococcus aureus is a leading cause of nosocomial and community-acquired infections, and the appearance of antimicrobial resistance continually presents new treatment challenges. In addition, S. aureus is a biofilm-producing pathogen that is commonly implicated in implant-associated infections. Biofilm formation represents a unique mechanism by which S. aureus and other microorganisms are able to avoid antimicrobial clearance and establish chronic infections, and these infections are characteristically refractory to standard antimicrobial therapy. There is a great need for the development of effective animal models for the study of biofilm infections and novel therapeutics. There is also substantial interest in the utilization of noninvasive, in vivo data collection techniques to reduce animal numbers required for the execution of infectious disease studies. To address these needs, we evaluated three murine models of implant-associated biofilm infection using in vivo bioluminescent imaging (BLI) techniques. The goal of these studies was to identify the model that was most amenable to development of sustained infections which could be repeatedly imaged in vivo using BLI technology. We found that a subcutaneous (s.c.) mesh and a tibial intramedullary (i.m.) pin model both maintained consistent levels of bioluminescence for up to 35 days post-infection, with no implant loss experienced in either model. In contrast, a s.c. catheter model demonstrated significant incidence of incisional abscessation and implant loss by day 20 post-infection. The correlation of bioluminescent measurements and bacterial enumeration was strongest with the s.c. mesh model whereas the correlation was weaker with the i.m. pin model. These data suggest that the s.c. mesh model is the most appropriate animal model of the three evaluated for the prolonged study of biofilm infections using BLI. Vaccination has been proposed as a potential therapeutic strategy for chronic staphylococcal infections; however recent attempts to develop an effective vaccine have been met with marginal success. One of the most important virulence factors of S. aureus is the membrane-bound protein Staphylococcal Protein A (SpA), which functions to inhibit both the innate and adaptive immune responses of the host. The majority of clinically relevant strains of S. aureus express SpA, making this protein a natural target for novel immunotherapeutics. A nontoxigenic form of SpA was previously developed, and prophylactic immunization with the protein was shown to promote innate and adaptive immune responses that are protective against disease in a mouse model of S. aureus bacteremia. This recent discovery further suggests that neutralization of SpA may improve clinical outcomes of staphylococcal infection. In the present study, we sought to determine the value of therapeutic vaccination targeting SpA for treatment of S. aureus biofilm infections. Our findings demonstrated that mice treated with repeated SpA vaccination following subcutaneous placement of S. aureus-coated mesh implants did not exhibit improved bacterial clearance when compared with untreated mice, although a strong humoral immune response to vaccination was observed. Using in vivo bioluminescent imaging, we also showed that the bacterial burden remained consistent between the vaccinated and unvaccinated groups of animals over the course of the study period. Furthermore, in vitro assays demonstrated that antibodies against SpA did not bind effectively to S. aureus, however opsonophagocytic clearance of planktonic bacteria was enhanced in the presence of whole blood from immunized mice. While these results suggest that SpA vaccination was not an effective tool for the treatment of S. aureus biofilm infections, more research is necessary to determine the specific role of SpA in biofilm development and other non-SpA mechanisms that are responsible for biofilm resistance.Item Open Access Promoting psychosocial health and empowerment among female sex workers in Nepal: a pilot peer education intervention(Colorado State University. Libraries, 2016) Menger, Lauren Marie, author; Stallones, Lorann, advisor; Fisher, Gwen, committee member; Kaufman, Michelle, committee member; Ryan, Elizabeth, committee memberAcross contexts, female sex workers (FSWs) may be exposed to varying degrees and combinations of risks in their work, including but not limited to long hours, poor working conditions, disease transmission, unplanned pregnancy, violence, drug and alcohol use and abuse, debt, and various forms of harassment, discrimination, and exploitation. It is likely that the risks associated with sex work are greater in developing countries where sex workers have a low and stigmatized status, minimal capacity to earn an adequate income, limited level of control regarding clients accepted and services rendered, and restricted access to sufficiently resourced health and other services and support structures. The sex industry in Nepal is synonymously referred to as the entertainment sector. Women in the entertainment sector (WES) in Nepal are vulnerable to an array of occupational risks, which compromise their psychosocial health and empowerment, in turn limiting their ability to thrive and engage in protective behaviors. The present study involved the pilot test of a peer education intervention in collaboration with a non-governmental organization (NGO) to empower and promote the psychosocial and occupational health of WES in Kathmandu, Nepal. Ten WES were trained as peer educators (PEs) and, through formal and informal teaching opportunities, reached over 140 FSWs with psychosocial health promotion messages. In addition to a detailed literature review, method, and discussion, this dissertation comprises three manuscripts. The first manuscript presents results from a quasi-experimental pre/post evaluation with 160 WES, including those who were (n = 96) and were not (n = 64) exposed to the PEs, to assess the impact of the program on psychosocial and occupational health and empowerment outcomes. Results indicate that WES who were exposed to the psychosocial health promotion messages of the PEs reported significantly improved psychosocial health knowledge and perceived self-efficacy, ability to access resources, happiness, and job control compared to WES who were not exposed to the PEs. The second manuscript presents results from a mixed-methods evaluation to assess the feasibility of the program and its impact on the psychosocial and occupational health and empowerment of the 10 WES trained as PEs. PEs were surveyed at baseline, immediately post intervention, after 2-months, and after 10-months to evaluate psychosocial and occupational health, empowerment, and peer education efficacy. Upon completion of the program, one-on-one exit interviews were conducted with nine of the PEs and two field staff from the partner NGO to solicit more in-depth feedback about the program. PE survey results indicate the program had a significant impact on some aspects of psychosocial health and empowerment, with positive trends on many other variables. Exit interviews revealed additional positive impacts of the program, including enhanced confidence and communication skills and increased self-awareness and self-care behaviors. Overall, the findings presented in these two manuscripts suggest peer education is both a feasible and promising means to enhance the psychosocial and occupational health and empowerment of WES in Nepal. The third manuscript details the processes implemented in this pilot study. Peer education methods have been established as a promising way to reach FSWs and other vulnerable and hard-to-reach populations with health promotion programming; however, there is scant published information about how such programs are designed and implemented. This lack of process information contributes to poor clarity regarding how to effectively develop and execute peer education programs and increases the propensity for repetition among failed strategies. Using this pilot program as an example, this manuscript offers an in-depth vantage point into the black box of peer education by outlining the specific steps taken while designing, implementing, and evaluating the program. While considering each phase of the project, the challenges encountered along the way as well as the effective strategies implemented to overcome them are reviewed, with a focus on offering practical tips and strategies. Conclusions and a summary of recommendations for those interested in implementing similar programs are discussed. These three manuscripts as a whole can be used to inform future interventions aiming to enhance the psychosocial and occupational well-being and empowerment of sex workers and other vulnerable and hard-to-reach working populations through peer education methods.Item Open Access Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses(Colorado State University. Libraries, 2016) Hu, Shuang, author; Akkina, Ramesh, advisor; Quackenbush, Sandra, committee member; Aboellail, Tawfik, committee member; Ryan, Elizabeth, committee memberLentiviral vector system is widely used in gene therapy. Although the envelope glycoprotein of vesicular stomatitis virus (VSV-G) has been mostly used to pseudotype lentiviral vectors, its disadvantages such as low transduction levels in certain cell types and sensitivity to inactivation by human complement hinders the usage of VSV-G pseudotyped lentiviral vectors in some cells or its direct in vivo clinical application. Aiming at overcoming some of these drawbacks of VSV-G, we evaluated two novel vesiculovirus envelope glycoproteins from Chandipura virus and Piry virus (CNV-G and PRV-G), as alternatives to VSV-G. Our results showed that pseudotyped lentiviral vectors could be generated with both these envelopes with high titers and stabilities similar to VSV-G. While displaying a more selective tropism than VSV-G, both CNV-G and PRV-G pseudotypes were found to be efficient in transducing a variety of cell types that include neuronal, fibroblastic and epithelial cells from across different species in addition to a number of human T-lymphocyte cell lines in vitro. Additionally, both the novel pseudotypes were found to be more resistant to human sera inactivation than the VSV-G pseudotype, thus providing better candidates for systemic administration. These data, taken together, establish that both Chandipura and Piry viral glycoproteins are suitable alternative candidates for lentiviral vector pseudotyping with an additional advantage for potential in vivo use in various gene therapy-based applications.Item Open Access The role of vitamin A and mycobacterial lipids in the immunopathogenesis of Mycobacterium tuberculosis infections(Colorado State University. Libraries, 2023) Harris, Macallister C., author; Podell, Brendan, advisor; Zabel, Mark, committee member; Henao Tamayo, Marcela, committee member; Ryan, Elizabeth, committee member; Basaraba, Randall, committee memberUntil recently Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), was the deadliest and most ubiquitous infectious disease in the world, infecting an estimated 1.7 billion people as of 2018 and killing 1.6 million people in 2021. However, recently COVID-19 has displaced this disease as the most fatal disease, and in the process of doing so has disrupted integral structural apparatuses meant to monitor and treat TB in endemic countries. This degradation has led to an unprecedented rise in disease transmission with a 4.3% rise in infection, with 10.6 million new infections in 2021. Further compounding the problem of this wave of Mtb infections is the rise of multidrug resistant infections, accounting for 3.9% of all new infections and responsible for 14% of Mtb fatalities in 2017. With an upsurge in case incidence, faltering antibiotic regimens and a variably effective 100-year-old vaccine, there is a new push in understanding disease comorbidities and alternative treatments for TB. Although over 1 billion people are presumed to be infected with Mtb, though not all of these patients have clinical TB. In fact, the WHO estimates that 1/3 of infections results in latent disease, a non-clinical phase of the infection which can last in definitively or eventually progress into active, clinical apparent TB. Patient comorbidities have become an intense area of study for further defining the delineating factors that direct infected individuals into either active infection, latent infection, or overcoming the infection. Two defining comorbidities, HIV status and diabetes have been correlated to an increased risk for having progressive TB. HIV infections in itself has been demonstrated to cause a 20 to 30 fold increase risk in clinical manifestations of a Mtb infection, leading to 187,000 deaths in 2021. An equally prolific area of Mtb research lies in developing new, efficacious prophylactic and active infection treatment regimens. As previously mentioned, current first line drugs regimens, such as rifampicin and isoniazid, are faltering against multidrug resistant Mtb. Additionally, the only widely available vaccine, the Bacillus Calmette-Guerin vaccine, has high efficacious against several forms of TB, like adolescent TB meningitis, but has variable to low efficacy in control of classic pulmonary TB. These factors are driving research of novel treatments and vaccines that utilize niche characteristics of Mtb, host immunity and overall TB disease pathogenesis to prevent clinical disease manifestation. This thesis centers at the intersection of these two areas of this immunologic research, Mtb comorbidities and investigating a novel branch of immunology, by investigating the immunopathogenesis of TB in relation to a newly recognized comorbidity, vitamin A deficiency, and examining the utility of harnessing a niche lipid-based branch of immunology, CD1 lipid restricted immunology, to combat TB disease progression. In chapter one, this thesis, stemming from the foundational epidemiological work of Dr. Megan Murray, endeavored to investigate the pathophysiology of vitamin A deficiency and TB in a highly translatable guinea pig model. This novel vitamin A animal model demonstrated that vitamin A deficiency leads to a dysregulated immune system with atypically granulomatous lesions, skewed inflammatory population and contradicting inflammatory gene profiles. These changes lead to progressive clinical disease with increased pulmonary bacterial burden and splenic lesion burdens. Additionally, this chapter was able to demonstrate the ability to partially alter the detrimental effects of vitamin A deficiency during the course of an active Mtb infection via reintroduction of a vitamin A sufficient diet. In chapter two, this thesis aimed to establish the kinetic expression of CD1 glycoproteins, the lipid antigen presenting molecule homologous to MHC I and the linchpin to lipid-restricted immunology, within tissues of infected guinea pigs. This work demonstrated CD1b upregulation during the early adaptive immunology phase of infection by profiling of CD1 glycoproteins via flow cytometric analysis, immunohistochemical tissue evaluation, and CD1 specific qPCR profiling. This study went further by not only establishing kinetic expression of this molecule but correlating that expression back to a functional cytolytic response driven by CD1-restricted T cells. Lastly, chapter three of this thesis aimed to build on the CD1 work in chapter two by assessing the role CD1 lipid restricted immunity plays in the disease pathogenesis of Mtb infections. To perform this work CD1 glycoprotein invivo modulation techniques were perfected via the use of CD1 ortholog specific synthetic antisense RNA, morpholinos, to down regulate CD1 protein translation. To stimulate CD1 expression upregulation, a pleotropic growth factor, FLT3-L, previously shown to upregulate CD1, was utilized. This chapter demonstrated that these compounds were able to alter CD1 expression in vivo when evaluated via flow cytometric analysis, immunohistochemically, and via altered qPCR profiles. Additionally, this chapter showed the ability to hamper the functional cytolytic activity of CD1-restricted T cells via CD1 down regulation. Although CD1 glycoprotein expression was altered, disease progression of Mtb in the guinea pig, as measured by lesion and bacterial burden, remained unaltered. Collective these aims serve as the beginning investigative studies into two unique and potential promising arenas of translational Mtb research. The discovery that vitamin A resupplementation mitigates vitamin A deficiency within chapter 1 has launched a host of different investigations examining the ability of this essential micronutrient to not only prevent clinical TB but to also serve as an immunologic boost to enhance the efficacy of a variety of Mtb treatment regimens. Chapters 2 and 3 reexamined CD1 immunity and the utility of the guinea pig for lipid immunologic work. Though much work has yet to be done in this niche branch of immunology, these chapters lay the ground work and provide the new tools for future studies into potential efficacious and translatable lipid-based vaccination schemes. Tuberculosis, caused by Mycobacterium tuberculosis, is a disease with global ramifications that requires innovative research to meet the new challenges on the horizon. This dissertation looked to meet those challenges by contributing new knowledge to two developing fields of immunologic research while also laying out the tools for future groundbreaking work.