Hemorrhagic enteritis is a disease of turkeys caused by virulent strains of Turkey Hemorrhagic Enteritis Virus (THEV) resulting in depression, splenomegaly, intestinal hemorrhage, immunosuppression, and mortality. Avirulent strains that do not produce intestinal lesions and mortality are used in live-virus vaccines that protect turkeys from virulent field challenge. The cause for the difference in phenotype between virulent and avirulent strains is unknown. The full-length genome of the Virginia Avirulent Strain (VAS) of THEV was sequenced and compared to the genome sequence of a virulent field isolate from Israel. Genetic differences were found in seven viral genes. Further sequencing narrowed the focus from seven genes to three: ORF1, E3, and Fiber. Consistent variation in these genes between strains of THEV with different phenotypes strongly indicates these genes as key factors affecting virulence. THEV is an officially recognized member of the viral family Adenoviridae, genus Siadenovirus. The genomes of the members of the genus, THEV and Frog Adenovirus 1, are not well-characterized. The genome sequences of both members were compared for the prediction of genetic and structural elements. Common features were found that distinguish this genus from all other adenoviruses, and differences were found that possibly contribute to host specificity of the members. The VAS is known to stimulate a life-long protective antibody response, though viral replication is only of short duration. Several studies were undertaken to determine changes in virus location and serology over time. Viral DNA was detected in various tissues through 15 weeks post-infection in the presence of high antibody titers. THEV infection was found to be similar to the non-lytic persistent infections seen with human adenoviruses. Regardless of the mechanism involved in the persistent stimulation of antibodies in infected turkeys, the VAS was shown to be an ideal vector for use in a recombinant live-virus vaccine. The next step in THEV research should be the creation of a full-length infectious DNA clone, which could be used in the creation of a recombinant vaccine. The infectious clone would also allow for the systematic testing of genes that are suspected to be involved in virulence. / Ph. D.

<p> Background: Vitamin D activity is important for the functioning of a broad range of body systems. Some of these, including the skeletal, immune, and cardiovascular systems, are particularly relevant in the management of HIV-infection; thus, and in consideration of evidence that factors associated with the scenario of HIV-infection can disrupt vitamin D metabolism, the assessment of vitamin D status in people living with HIV-infection may be particularly important. In this thesis, I address cardiovascular implications of vitamin D status in HIV-infection. More specifically, and based on a growing body of evidence implicating low vitamin D status in the development of cardiovascular disease (CVD), I hypothesized that in HIV-positive adults low 25-hydroxyvitamin D (25(0H)D) concentration would be associated with increased subclinical vascular disease as measured by carotid intima-medial thickness (IMT).</p> <p> Methods: Using regression analyses I cross-sectionally studied the relationship between 25(0H)D and carotid IMT in 283 participants of the Canadian HIV Vascular Study, a prospective study of CVD risk among HIV-positive Canadians.</p> <p> Results: The prevalence of vitamin D deficiency in the Canadian HIV Vascular study was surprisingly low. Plasma 25(0H)D by quartile was not associated with carotid IMT. However, in restricted cubic spline regression analyses designed to accommodate non-linearity there was evidence of an inverted U-shaped 25(0H)D-carotid IMT relationship. In exploratory regression models restricted to participants comprising the suboptimal range of vitamin D status, lower 25(0H)D concentration was statistically significantly associated with lower carotid IMT after adjustment for known CVD risk factors and other variables hypothesized to potentially confound a 25(0H)D-carotid IMT association.</p> <p> Main implication: While inference from these exploratory findings requires cautious interpretation, future investigations into the relationship between vitamin D status and vascular disease should consider the problem of non-linearity as a feature of primary analyses; otherwise, such studies might fail to detect a true association.</p> / Thesis / Master of Science (MSc)

Infection & Immunity / Currently, there are 38 million people living with human immunodeficiency virus (HIV-1) worldwide and there were 680,000 HIV-related deaths in 2020 alone. The greatest cause of mortality in people living with HIV (PLHIV) is infection with opportunistic pathogens such as tuberculosis (TB), which accounts for one third of HIV-related deaths. PLHIV are 20 times more susceptible to TB and co-infection leads to significantly worsened outcomes in terms of both diseases. Humanized mouse (hu-mouse) models, which possess human immune cells for HIV to infect, have been useful for HIV research. Our aim is to create hu- mouse models of HIV, TB and co-infection to investigate disease progression, immune responses, therapeutics, prevention and vaccination. NOD-Rag1null-IL2rgnull (NRG) mice are highly immunocompromised mice that are traditionally used to generate hu-mouse models. We are also developing NRG mice that are transgenic for human HLA-DR4 and HLA-A2 (DRAG-A2) and similar mice have been reported to have improved immune responses. NRG and DRAG-A2 mice were humanized with hematopoietic stem cells obtained from human umbilical cord blood. DRAG-A2 mice had significantly higher engraftment success rates (defined as the percentage of mice with >10% hCD45+) as well as higher overall CD45+ leukocyte, CD4+ T cell, CD19+ B cell and CD14+ monocyte reconstitution in the blood compared to huNRGs. huNRG mice were permissive to infection with JR-CSF or NL4.3-Bal-Env HIV-1 intravaginally or systemically. huDRAG-A2 mice were also infected intravaginally with NL4.3-Bal-Env HIV-1. huDRAG-A2 mice, but not huNRGs, produced HIV-specific IgG, indicating improved immune responses. huNRG mice were infected intranasally with mCherry-Erdman, YFP-H37Rv or H37Rv Mtb. huDRAG-A2 mice were also infected with H37Rv. Human immune cell involvement and human-like granuloma formation was observed using flow cytometry and immunohistopathology. These findings show that the DRAG-A2 model may be optimal for investigating HIV, TB and co-infection, which continue to be serious global health concerns. / Thesis / Master of Science (MSc) / Human immunodeficiency virus (HIV) and tuberculosis (TB) are infectious diseases that affect millions of people worldwide every year. The greatest cause of death in people living with HIV is co-infection with TB and HIV-positive individuals are much more likely to get TB. Humanized mouse (hu-mouse) models possess human immune cells for HIV to infect and are useful for studying HIV. Our goal is to create hu-mouse models of HIV, TB and HIV/TB co-infection that will allow us to study how these diseases interact. We are currently developing a traditional hu-mouse model (known as NRG), as well as an improved next-generation model (known as DRAG-A2) with a more functional immune system. Both models have been successfully infected with HIV or TB. Only DRAG-A2 mice were able to make antibodies against HIV. The improved DRAG-A2 model will enable future studies on HIV, TB and co-infection, which continue to be understudied global problems.

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

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