Human metapneumovirus (HMPV) is a respiratory pathogen in the Paramyxoviridae family that infects nearly 100% of the world population. This enveloped RNA virus causes severe viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide. Despite its prevalence and importance to human health, no therapies are available against this pathogen. Entry of paramyxoviruses into host cells generally requires the coordinated activity of the attachment glycoprotein, G, which interacts with a cell receptor, and the fusion glycoprotein, F, which promotes subsequent fusion of viral and cellular membranes. However, HMPV F is the primary viral protein mediating both binding and fusion for HMPV. Previous work that showed HMPV F mediates attachment to heparan sulfate proteoglycans (HSPGs), and some HMPV F fusion activity can be promoted by acidic pH. The work presented here provides significant advances in our understanding of the fusion and binding events during HMPV infection. We demonstrated that low pH promotes fusion in HMPV F proteins from diverse clades, challenging previously reported requirements and identifying a critical residue that enhances low pH promoted fusion. These results support our hypothesis that electrostatic interactions play a key role in HMPV F triggering and further elucidate the complexity of viral fusion proteins. Additionally, we characterized the key features of the binding interaction between HMPV and HSPGs using heparan sulfate mimetics, identifying an important sulfate modification, and demonstrated that these interactions occur at the apical surface of polarized airways tissues. We identified differences in particle binding related to the presence or absence of the HMPV G and SH glycoproteins. Lastly, we characterized paramyxovirus infection in cystic fibrosis bronchial epithelial cells, identifying a potential specific susceptibility to HMPV infection in these individuals. The work presented here contributes to our understanding of HMPV infection, from mechanisms of early events of entry to clinical scenarios.

Research has shown that maximal exercise has a significant effect on cells of the immune system. Specifically, lymphocyte count increases during exercise and decreases to a value lower than baseline following an acute exhaustive bout of exercise. The overall lymphocyte response is well characterized, however, the ability of exercise to affect lymphocyte subfractions is unknown to our knowledge. The purpose of this study was to assess and evaluate the affects of a simulated tennis match across two sessions on lymphocyte subsets. Initial measurements such as age, height, weight, skinfold analysis, and heart rate were recorded for each player, as well as blood samples being obtained by a finger prick before and after the tennis sessions. The tennis protocol started with five serves to the deuce court and five serves to the ad court, then individuals hit twenty-four forehands and twenty-four backhands against an oscillating ball machine. Each bout of serves and ground strokes were repeated ten times, with one minute rests in between each session. Before and immediately after completing the tennis trial, subjects were pricked with a lancet on the non dominant hand so to obtain at least two capillary tubes of blood. Whole blood was then added to the antibody cocktail, which is mixed according to the antibodies that were tested, which were CD4, CD8, CD19, CD95, and CX3CR1. Whole blood was added to red blood cell lysis buffer and fixation buffer, and the blood solution was incubated with antibodies specific to cell phenotype. The main results of this study indicated that there was a decrease in mainly post cell counts in pre and post CD19/CD95 measurements (P= .007), an increase in CD8/CX3CR1 in pre counts and an increase then decrease in post counts without wearing the bionic glove (P= .042), and a decrease in CD4 in the post count measurement with the bionic tennis glove (P= .043). The study’s can assist in making recommendations for after match treatment such as health and diet suggestions. Knowledge of prevention and treatment methods are low in the field of tennis and immune functions, so findings in this area could prevent elite athletes from contracting infections between matches.

Necroptosis, a type of regulated necrotic cell death, involves cell membrane permeabilization and has been implicated in various acute and chronic pro-inflammatory diseases, including ischemia-reperfusion injury and neurodegenerative diseases. By using in vitro reconstitution studies and a chemical inhibitor, the kinase activity of the serine/threonine kinase RIPK1 had been shown to regulate necroptotic signaling downstream of TNF and Toll-like receptors (TLRs). To investigate the contribution of RIPK1 kinase activity to inflammation and necroptosis in vivo, we generated kinase inactive RIPK1 knock-in mice. Utilizing fibroblasts and macrophages from these mice, we demonstrate that RIPK1 kinase activity is required for necroptotic complex formation and death induction downstream of TNFR1 and TLRs 3 and 4. We show that RIPK1 kinase inactive mice are resistant to TNF-induced shock and exhibit impaired upregulation of TNF-induced cytokines and chemokines in vitro and in vivo. By using bone marrow reconstitution experiments, we demonstrate that RIPK1 kinase activity in a non-hematopoietic lineage drives TNF-induced lethality. We establish that RIPK1 kinase activity is required for TNF-induced increases in intestinal and vascular permeability and clotting, and implicate endothelial cell necroptosis as an underlying factor contributing to TNF/zVAD-induced shock. Thus, work in this thesis reveals that RIPK1 kinase inhibitors may have promise in treating shock and sepsis.

The purpose of this investigation was to observe changes induced by types 1 and 2 herpes simplex viruses (HSY 1 and HSY 2) when these viruses were cultivated in fish 2 cells. The possibility of attempting to use human strains of herpesviruses in order to transform normal fish tissue culture preparations was considered. Three different fish cell cultures were tested: CHSE-214 cell lines were derived from embryonic chinook salmon (Oncorhynohus tshawytscha), and STE-137 cell lines were obtained from embryonic steelhead trout (Salmo gairdneri). Cells isolated from immature ovaries of rainbow trout (Salmo gairdneri) were propagated by the investigator in these studies. Both HSV 1 and HSV 2 demonstrated cytopathic effects on the 3 different cell types described above when these cells were observed during the exponential growth phase. If virus was added before cells were dividing actively, or when the cells were maintained in a stationary phase of growth, no effect was observed. Several methods were employed in an attempt to trans- form fish cells grown in tissue culture. Different quantities of live virus, preparations of ultraviolet light in- activated virus and purified viral DNA were used in these studies but malignant conversion by known viral components was not achieved. In these experiments, the 2B strain of cells obtained originally from immature ovaries of rainbow trout appeared to undergo spontaneous transformation thereby becoming established as a permanent cell line. To date tissue cultures designated 2B have undergone 25 transfers in this laboratory.

Phenylethanolamine-N-methyltransferase (Pnmt) is the enzyme that catalyzes the conversion of noradrenaline to adrenaline. These catecholamines are synthesized in the medulla of the adrenal gland and by some neurons of the central nervous system. The precise location of Pnmt action in the brain and its physiological significance are unknown. Prior studies led by Aaron Owji, a graduate student in Dr. Ebert’s laboratory, showed that mice with selectively ablated Pnmt cells show signs of neurological defects such as abnormal gait, weakened grip strength, lack of balance, reduced movement, and defective reflexes during tail suspension tests. The cerebellum is a small section of the brain that is responsible for fine-tuning motor commands. Since the Purkinje cells of the cerebellum act as the sole source of output from the cerebellar cortex, impairment of these cells could possibly account for the motor deficits seen in the mice models. The purpose of this project is to determine if there is indeed a change in Purkinje cells between wild type mice and Pnmt-ablated mice. The first aim is to identify quantitative differences in cell count between both genotypes. The second aim is to determine any morphological changes in the Purkinje cells. The main technique used in this project is immunohistochemistry in which cerebellum tissue from mice models are stained with Calbindin (a cellular marker for Purkinje neurons) and imaged with a confocal microscope. Results showed a slight reduction in the Purkinje cells of the ablated mice compared to the control genotype, accompanied with observable differences in cell structure. Understanding catecholamine pathway mechanisms in the nervous system is imperative for elucidating and targeting key players in neurodegenerative disorders.

Human immunodeficiency virus (HIV) has become a global pandemic over the past few decades, with new infections and related deaths in the millions each year. There is no cure in sight for HIV-1 infection, and there has been little progress in developing an efficacious vaccine. Heterosexual transmission of HIV-1 remains the principal mode of transmission throughout the world and thus measures, such as topical vaginal microbicides, to prevent infection of the female reproductive tract are actively being explored. Recent trials of topical vaginal microbicides have shown that their interaction with the mucosal surfaces of the female reproductive tract as well as semen can hinder microbicide effectiveness against HIV-1 infection. Therefore, understanding the role these fluids play in HIV transmission would be critical towards developing effective antiviral prophylaxes. A recent study from our group demonstrated that human cervicovaginal secretions contained numerous cationic antimicrobial peptides and proteins, which collectively inhibited HIV-1 infection of target cells and tissues. To ascertain if human seminal plasma (SP), the main vector responsible for transmitting HIV-1, exhibited antiviral activity we utilized several antiHIV assays in the presence or absence of minimally manipulated SP. The majority of the intrinsic anti-HIV-1 activity of SP resided in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multi-step chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP. Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" capable of enhancing HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity. Our studies demonstrate the overall antiviral activity of human SP, but there is still a critical need for effective topical vaginal microbicides that can prevent HIV-1 transmission. The synthetic human retrocyclins are cyclic antimicrobial peptides that are remarkably active against HIV-1, and are being developed as topical vaginal microbicides. Herein, we assessed whether the putative proviral SEVI was able to adversely affect the anti-HIV-1 activity of the retrocyclin analog RC-101. While SEVI alone enhanced viral infection, this effect was completely negated in the presence of RC-101. Retrocyclins such as RC-101 are inhibitors of HIV-1 entry, by preventing gp41-mediated viral fusion. Interestingly, using an HIV-1 reverse transcriptase (RT) specific assay, we also determined that RC-101 directly inhibited the activity of RT in a dose dependent manner, suggesting a secondary mechanism of viral inhibition. Our group has determined that RC-101 induces only a modest level of resistance in HIV, which may be due in part to RC-101's dual mechanisms of viral inhibition.

Ebola virus (EBOV) is a re-emerging zoonotic virus (it is transmitted between animals and humans) that causes acute hemorrhagic fever and a high fatality rate in humans. First reported in 1976 in the Democratic Republic of the Congo (formerly Zaire), the virus is transmitted between humans through direct contact with body fluids of an infected person, causing fever, weakness, diarrhea, abdominal pain, cramping, nausea and vomiting in those affected. There is neither a licensed vaccine nor an approved treatment for Ebola virus in human patients. The reservoir species for Ebola virus is similarly unknown, as many studies have attempted yet failed to isolate living virus from potential candidates. The widely accepted and circulated hypothesis based on preliminary findings of outbreaks past is that bat species, specifically the fruit bat species Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquata are potential reservoirs. Recent reports, especially concerning findings from the 2014 Ebola outbreak, have determined that insectivorous bats could similarly be reservoir species. Successful isolation of a live virus from a bat species found through a widened sampling of a variety of bat species would confirm the hypothesis that bats, either fruit or insectivorous, are the reservoir species for Ebola virus.

The advent of high-throughput sequencing has brought about the creation of an unprecedented amount of research data. Analytical methodology has not been able to keep pace with the plethora of data being produced. Two assays, ImmunoSEQ and the cytokinesisblock micronucleus (CBMN), that both produce count data and have few methods available to analyze them are considered. ImmunoSEQ is a sequencing assay that measures the beta T-cell receptor (TCR) repertoire. The ImmunoSEQ assay was used to describe the TCR repertoires of patients that have undergone hematopoietic stem cell transplantation (HSCT). Several different methods for spectratype analysis were extended to the TCR sequencing setting then applied to these data to demonstrate different ways the data set can be analyzed. The different methods include CDR3 distribution perturbation, Oligoscores, Simpson's diversity, Shannon diversity, Kullback-Liebler divergence, a non-parametric method and a proportion logit transformation method. Herein we also demonstrate adapting compositional data analysis methods to the TCR sequencing setting. The various methods were compared when analyzing a set of 13 subjects who underwent hematopoietic stem cell transplantation. The eight subjects who developed graft versus host disease were compared to the five who did not. There was no little overlap in the results of the different methods showing that researchers must choose the appropriate method for their research question of interest. The CBMN assay measures the rate of micronuclei (MN) formation in a sample of cells and can be paired with gene expression or methylation assays to determine association between MN formation and other genetic markers. Herein we extended the generalized monotone incremental forward stagewise (GMIFS) method to the situation where the response is count data and there are more independent variables than there are samples. Our Poisson GMIFS method was compared to a popular alternative, glmpath, by using simulations and applying both to real data. Simulations showed that both methods perform similarly in accurately choosing truly significant variables. However, glmpath appears to overfit compared to our GMIFS method. Finally, when both methods were applied to two data sets GMIFS appeared to be more stable than glmpath.

A recent study using human fecal samples in conjunction with a culture-independent approach revealed immense diversity of antibiotic resistance (AR) genes in the human gut microflora. We hypothesize that food animal gut microflora also contain diverse and novel AR genes which could contribute to the emergence and transmission of AR in pathogens important in animal and human health. To test this, we examined AR reservoir in chicken gut microflora using a metagenomic, functional cloning method. Total genomic DNA was extracted from individual cecal contents of two free range chickens and two conventionally raised chickens. The DNAs were physically sheered into 1 to 3 kb fragments, cloned into expression vector pZE21-MCS, and transformed into E. coli TOP10 host strain, resulting in four metagenomic libraries of a total size of 108 base pairs per library. The AR transformants from the libraries were selected on plates containing the specific antibiotic of interest; six antibiotics including ampicillin, tetracycline, chloramphenicol, spectinomycin, ciprofloxacin and norfloxacin were used for screening. Plasmids from selected transformants were extracted and subjected to sequence analysis of inserted fragments. Identified AR genes were annotated and aligned with homologs that have been deposited in GenBank. A total of 12 AR genes and 3 AR genes were identified from the microbiome in conventionally raised chickens and free-range chickens, respectively. Of the identified 15 AR genes, 8 genes that confer resistance to ampicillin, spectinomycin or chloramphenicol shared low sequence similarity (58% - 76% at amino acid level) with the corresponding AR genes previously identified using culture-dependent approaches. Notably, among the 8 novel AR genes identified in this study, 4 genes also shared low sequence similarities (59%-76% at amino acid level) with recently identified AR genes in human gut. An E. coli-Campylobacter shuttle vector bearing the flaA sigma 28 promoter was constructed. Two novel genes conferring resistance to ampicillin (FRAmp1.1) and spectinomycin (FRSpe1.1) were cloned into this new expression vector, respectively. The derived vectors have conferred increased AR in C. jejuni, a leading zoonotic bacterial pathogen causing human gastroenteritidis in many industrialized countries. Together, findings from this study showed the effectiveness of the metagenomic approach for examination of AR reservoir in food animals, revealed novel AR resistance genes in chicken gut microflora, and demonstrated the functionality of such AR genes in foodborne human pathogens.

Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Disease (MCTD) are chronic, autoimmune disorders that target overlapping autoantigens and exhibit similar clinical manifestations. Despite 40 years of research, a reliable biomarker capable of diagnosing these syndromes has yet to be identified. Previous studies have confirmed that components of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) such as U1A are 1000 fold more autoantigenic than any other nuclear component in SLE patients. Based on these findings, I hypothesize that models derived from the U1 snRNP autoantigenic properties could distinguish SLE from MCTD patients. To test this hypothesis, 30 peptides corresponding to protein regions of the U1 snRNP were tested in triplicates by indirect ELISA in sera from SLE or MCTD subjects. In addition laboratory tests and clinical manifestations data from these patients were included and analyzed in this investigation. Statistical classification methods as well as bioinformatics pattern recognition strategy were employed to determine which combination, if any, of all the variables included in this study provide the best segregation power for SLE and MCTD. The results confirmed that the IgM reactivity for U1 snRNP and U1A have the power to significantly distinguish SLE from MTCD patients as well as identify kidney and lung malfunctions for these subjects (p ≤ 0.05). Furthermore, the data analysis revealed eight novel classification rules for the segregation of SLE and MCTD which are a better classification tool than any of the currently available methods (p ≤ 0.05). Consequently, the results derived from this study support that SLE and MCTD are indeed separate disorders and pioneer the description of eight novel classification criteria capable of significantly discerning between SLE and MCTD patients (p ≤ 0.05).