From Micro To Macro: Examining Potential Microbiome Mediated Influences On Human Growth And Health Outcomes Through Breastfeeding And Antibiotic Exposures, Nicole K. Phillips

Electronic Thesis and Dissertation Repository

Human microbiome research has rapidly developed over the past two decades yet absent from most research is the composition and dynamics of microbiomes within human populations. Given the limitations in longitudinal studies which requires decades of repeated microbe taxonomic testing of a population sample, an alternative option is to examine microbiomes and their influences via proxies using pre-existing health datasets. This research demonstrates preliminary associations between presumed disrupted and supportive microbiomes dynamics proxied by antibiotic and breastmilk exposure respectively. Using health record data across the life span from approximately 500,000 U.K. participants, this research demonstrates variable altered growth and health …

A Workflow To Analyze Ethcd Mass Spectrometry Data For Studying Hiv Gp120 Glycosylation, Yingxue Sun

Electronic Thesis and Dissertation Repository

The great heterogeneity of HIV populations and richness of surface glycan clouds makes it difficult to locate a conserved and exposed protein epitope as an effective vaccine target. However, more than 80% new infections result from single transmitted founder (T/F) viruses. We set out to design a workflow to study the traits of T/Fs that allow for their superior infectivity, specifically, the glycosylation patterns of gp120, a subunit of HIV envelope protein responsible for binding to host cell receptors. Our main research methods include Western blot and mass spectrometry. Our current understanding of the mass spectrometry data indicates that our …

Encountering Lectins In The Recipient Mucosa: Implications Of N-Linked Glycosylation On Hiv-1 Transmission, Adam Meadows

Electronic Thesis and Dissertation Repository

Although several studies have determined key differences in envelope motifs between TF and chronic HIV-1, it is still not known what the overall glycosylation profile is that is selected for in a transmission event, as well as what contributes to this selection. Using a bottom-up approach of modifying specific viruses, determining their transmission fitness in an ex vivo tissue explant assay, and determining their glycan content, we have laid the basis for determining the overall glycan structure which is selected for in TF HIV-1. Preliminarily, we have shown that C-type lectins represent a stringent barrier to transmission and have several …

Endogenous, Controlled Expression Of Anti-Hiv-1 Broadly Neutralizing Antibody, Darshit Patel

Electronic Thesis and Dissertation Repository

Recently, researchers have identified a number of anti-HIV broadly neutralizing antibodies (bNAbs), such as VRC01 and N6, capable of targeting a broad range of HIV-1 strains. Passive immunization using these patient-derived bNAbs could provide temporary protection but are limited by the short antibody half-life. While current gene transfer technology allows sustained bNAb expression, it lacks the ability to control bNAb production in vivo resulting in possible autoimmunity. To address this issue of achieving controlled bNAb expression in vivo, we hypothesize that bNAb expression from transduced Flu-specific B cells can be activated and modulated by subsequent Flu immunizations in the …

Hiv-1 Group M Subtype Fitness, Disease Progression, And Entry Efficiency, Colin M. Venner

Electronic Thesis and Dissertation Repository

Human immunodeficiency virus type 1 (HIV-1) emerged in the human population shortly after the turn of the 19th century. Distribution of HIV-1 across the globe over the past 30–35 years can be traced to founder events with primordial HIV strains from sub-Saharan Africa. Even considering the burden of HIV in Africa, our knowledge of HIV-1 disease is still largely limited to subtype B HIV-1, a strain responsible for 3 million infections in North America and Europe as compared to the 33 million that are infected with HIV-1 subtypes A, C, D, and circulating and unique recombinant forms.

This dissertation analyzes …

Determining The Molecular Mechanisms Of Pacs-1-Mediated Protein Sorting, Brennan S. Dirk

Electronic Thesis and Dissertation Repository

Membrane trafficking events are required to direct proteins to their precise subcellular locations. The cellular Phosphofurin Acidic Cluster Sorting protein – 1 (PACS-1) has emerged as a protein of interest in controlling the localization of a multitude of cellular and viral proteins. Specifically, PACS-1 is hijacked by type-1 Human Immunodeficiency Virus (HIV-1) to contribute to immune evasion in addition to regulating neuroendocrine hormone storage and release. To accomplish this, PACS-1 connects the cytoplasmic tail of cellular receptors to the heterotetrameric adaptor proteins (APs) to form a functional trafficking unit. Throughout this dissertation, I explored the role of PACS-1 and AP-1 …

Interferon-Induced Herc5 Is A Novel And Potent Inhibitor Of Ebola Virus-Like Particle Production, Nina Hunt

Electronic Thesis and Dissertation Repository

Ebola’s severe pathogenesis can be attributed to its suppression of the Type I interferon (IFN) response, suggesting this pathway plays a role in restricting viral replication. One IFN-induced protein, HERC5, warrants further investigation as it inhibits the replication of evolutionarily diverse viruses. We showed that HERC5 drastically reduces the expression of structural protein VP40 at the protein and mRNA level. Mutagenesis of HERC5 demonstrated that the RCC-1-like domain is necessary and sufficient for restriction. This domain is also responsible for HERC5’s interaction with ZAP, a protein required for VP40 mRNA degradation. Finally, we showed that Ebola GP antagonizes HERC5 activity. …

The Role Of The Hiv-1 Accessory Proteins Nef And Vpu In Subverting Host Cellular Immunity, Emily N. Pawlak

Electronic Thesis and Dissertation Repository

The HIV-1 accessory proteins Nef and Vpu have no known enzymatic activity, yet these viral proteins are critical for HIV-1 viral spread and pathogenicity. To mediate this, these viral proteins bind to and hijack host cellular proteins, including proteins implicated in the membrane trafficking machinery. Key to the effects of these proteins is their ability to alter the subcellular localization of a plethora of cell surface receptors, including the key immune cell receptorsCD28 and MHC-I.However,the mechanism utilized by Nef and Vpu to modulate these proteins is not fully understood. Herein, we demonstrated that Nef and Vpu mediate a decrease in …

Understanding The Impact Of Hiv-1 Genetic Diversity On The Function Of Nef And Its Role In Serinc5 Antagonism, Aaron Leslie Johnson

Electronic Thesis and Dissertation Repository

HIV-1 Nef is a key pathogenic protein, allowing HIV-1 to evade the host immune system by downregulating MHC-I and CD4. Furthermore, it was recently discovered that Nef counteracts the host factor SERINC5 to increase HIV-1 infectivity, but the mechanistic details of the Nef:SERINC5 interaction still need to be explored. Throughout this dissertation, I will explore the hypothesis that the genetic diversity that defines HIV-1 has a pronounced effect on the HIV-1 protein Nef, altering its function between and within group M subtypes. To address this hypothesis I investigated how MHC-I and CD4 downregulation differ among all non-recombinant group M subtypes. …

The Surreptitious Survival Of The Emerging Pathogen Staphylococcus Lugdunensis In Macrophages Enhances S. Aureus Infection, David Watson

Electronic Thesis and Dissertation Repository

Staphylococcus lugdunensis is an opportunistic pathogen that can cause invasive infections suggesting an ability to circumvent host immunity. S. lugdunensis was shown to resist killing and persist within macrophages and acetylation of its peptidoglycan is important for this survival. This was consistent in vivo, as S. lugdunensis resides inside Kupffer cells for at least 16 hours post-infection in mice. Despite its capability for survival, S. lugdunensis is unable to replicate within phagolysosomes. Inhibiting phagolysosomal effectors allows S. lugdunensis to initiate replication, after which the bacteria escape phagosomal containment. Moreover, intracellular S. lugdunensis augments the growth S. aureus during co-infection. Eight …

Functional And Structural Mimicry Of A-Kinase Anchoring Proteins By Human Adenovirus E1a, Cason R. King

Electronic Thesis and Dissertation Repository

As an obligate intracellular parasite, human adenovirus (HAdV) must utilize host factors for survival and replication. Early during infection, its multifunctional E1A protein interacts with an impressive range of cellular target proteins to exert control over the cellular environment. Through these virus-host interactions, E1A massively reprograms both viral and cellular transcription to activate the other HAdV genes, downregulate the host’s immune response, and induce the cell cycle. Consequently, E1A converts the infected cell into a compliant state more amenable for HAdV replication, resulting from its numerous protein-protein interactions. I sought to examine E1A’s interaction with cellular protein kinase A (PKA), …

Identification And Characterization Of Farr And Fare As A Regulator And Effector Of Fatty Acid Resistance In Staphylococcus Aureus, Heba Alnaseri

Electronic Thesis and Dissertation Repository

Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions and in abscesses, specific mechanisms for regulating gene expression and intrinsic resistance in response to these fatty acids have not been reported. Through in vitro selection for increased resistance of S. aureus to linoleic acid, I identified fatty acid resistant clone FAR7, where a single nucleotide polymorphism caused a His₁₂₁Tyr substitution in an uncharacterized member of the TetR family of transcriptional regulators, which is divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multi-drug efflux pumps. I named these …

Characterizing The Interaction Between Human Adenovirus E1a And The Transcriptional Repressor Bs69, Ali Zhang

Electronic Thesis and Dissertation Repository

Protein products of the Early Region 1A (E1A) gene in human adenovirus 5 (HAdV-5) are the first viral proteins expressed upon adenovirus infection. E1A disrupts many cellular physiological events by binding to and regulating an impressive number of host factors. Of particular interest is BS69, a repressor of E1A transactivation. Due to the strong interaction observed between E1A and BS69, I hypothesize that these two proteins function together to disrupt gene expression within an infected cell.

Using in silico modelling and a series of yeast two-hybrid assays, I determined that residues 112-119 of HAdV-5 E1A is the minimal interacting region …

Investigating Adenovirus E1a As An Rna Polymerase Ii C-Terminal Domain Mimic And Its Role In Transcription Activation, Kristianne Jc Galpin

Electronic Thesis and Dissertation Repository

Viruses rely on host cell machinery, often mimicking cellular components, in order to circumvent host cell defenses and hijack cellular processes. DNA viruses, such as human Adenovirus (hAdV), rely on RNA Polymerase II (RNAPII) to transcribe viral genes. RNAPII has a C-terminal domain (CTD), made up of highly conserved heptad repeats of tyrosine-serine-proline-threonine-serine-proline-serine (YSPTSPS). Post-translational modifications of residues within the CTD, including phosphorylation, coordinates the transcription cycle. Several viruses, including Human Immunodeficiency Virus (HIV), Human Cytomegalovirus (hCMV), Epstein-Bar Virus (EBV) and Herpes Simplex Virus (HSV), modify the phosphorylation state of the RNAPII CTD by hijacking cellular cyclin dependent kinases (CDKs) …

Iron Acquisition Strategies Employed By Staphylococcus Lugdunensis, Jeremy R. Brozyna

Electronic Thesis and Dissertation Repository

Iron is crucial for many cellular processes including DNA synthesis and respiration. The majority of iron in mammals is in heme within hemoproteins, inside cells, or transported through circulation by the glycoprotein transferrin, which constitutes the greatest iron source in serum. Limiting iron availability is an important facet of nutritional immunity to help prevent infection.

Staphylococcus lugdunensis is a human skin commensal and opportunistic pathogen capable of causing a variety of infections, including particularly aggressive endocarditis. It is an emerging pathogen with elevated virulence compared to other species of coagulase-negative staphylococci. The versatility of S. lugdunensis to infect multiple niches …

Structural And Functional Studies Of The Heptose Modifying Enzymes That Play A Role In Campylobacter Jejuni Virulence., Heba Soliman Barnawi

Electronic Thesis and Dissertation Repository

Campylobacter jejuni is a major cause of gastroenteritis in humans. The capsule of some species contains unique modified heptoses. Heptose modification was elucidated for C. jejuni NCTC11168 and 18-176, and novel epimerases and reductases essential for the heptose modification were identified. We hypothesized that heptose modifying enzymes in C. jejuni have specific catalytic residues that allow for substrate and product specificity. Substrate synthesis, structural modeling, point mutations, and enzymatic analysis have been applied to map the active sites. Putative catalytic residues showed substrate and/or product specificity. The epimerases structures were solved by crystallography done by our collaborator. We also hypothesized …

Recombinase-Based In Vivo Expression Technology Identifies A Streptococcus Pyogenes Bacteriocin Important For Niche Adaptation In The Nasopharynx, Brent D. Armstrong

Electronic Thesis and Dissertation Repository

Streptococcus pyogenes is a Gram-positive, human-specific bacterial pathogen with the ability to cause a wide range of diseases from strep throat to necrotizing fasciitis and toxic shock syndrome. In addition, S. pyogenes may also induce post-streptococcal sequelae including rheumatic fever, acute glomerulonephritis, and reactive arthritis. Although primarily recognized as a pathogen, S. pyogenes also colonizes the skin and throat often without causing disease, and while numerous surface adhesions are important to attach to these surfaces, additional factors important for colonization and persistence by S. pyogenes are poorly understood. In addition to host defence mechanisms, the upper respiratory tract also contains …

Iron Acquisition Mechanisms And Their Role In Staphylococcus Aureus Survival And Virulence, Jessica R. Sheldon

Electronic Thesis and Dissertation Repository

To microbes and humans alike, iron is both essential, and potentially toxic, where homeostatic concentrations must be stringently maintained. Within the iron-restricted host, the survival and proliferation of microbial invaders is contingent upon exploiting the host iron pool. Staphylococcus aureus is a formidable pathogen, whose success is partly attributable to its multiple, and often redundant mechanisms for acquiring iron. A decade of research in staphylococcal iron acquisition has identified several uptake mechanisms, including, but not limited to, the elaboration of high-affinity iron scavenging siderophores (staphyloferrins A (SA) and B (SB)) and the expression of an iron-regulated surface determinant (Isd) pathway …

Clinical And Mechanistic Insights Into Novel Probiotic Functions And Formulations, Jordan Bisanz

Electronic Thesis and Dissertation Repository

Using a combination of hypothesis and discovery based approaches, the goal of this thesis was to better describe novel probiotic functions and their mechanisms while striving to better understand the effect of formulation on Bifidobacterium animalis subsp. lactis, Lactobacillus paracasei and L. rhamnosus.

Using RNA-Seq, a bacterial metatranscriptome analysis of a commonly consumed probiotic yogurt showed that the organisms adapted to storage time and flavor additions. This led to the discovery that in addition to the probiotic health benefits, members of the L. casei group (L. rhamnosus and L. paracasei) produce volatile sulfur compounds mediated by a novel …

The Role Of Streptococcus Salivarius As A Modulator Of Homeostasis In The Oral Cavity, Kyle W. Macdonald

Electronic Thesis and Dissertation Repository

The oral cavity contains many different surfaces all colonized by prokaryotes, of which over 700 have been identified. While almost all people have some degree of plaque formation, the more concerning diseases of caries, candidiasis and periodontal disease afflict many patients and represent a major public health concern. As these are all diseases which have a component attributable to parts of the microbiota, efforts to manipulate the microbes has until recently involved use of antimicrobial agents. However, due to side effects, resistance and failure to restore homeostasis, this approach is limited. As an alternative, the administration of beneficial microbes (probiotics) …

Hcpe, A Potential Immuno-Modulatory Protein From Helicobacter Pylori That Is Dependent On The Disulfide Bond Protein Dsbhp, Jeff Lester

Electronic Thesis and Dissertation Repository

H. pylori is a human gastric pathogen that colonizes ~ 50% of the world’s population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. H. pylori produces Helicobacter cysteine rich protein HcpE, a secreted protein which may play a role in virulence. In this study we show that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles, and may play a role in the modulation of H. pylori inflammatory responses. We identified that Dsb_HP is necessary for HcpE production and secretion in H. pylori, and …

Chemical Communication Of Antibiotic Resistance By Highly Resistant Bacteria., Omar M. El-Halfawy

Electronic Thesis and Dissertation Repository

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. I use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, I report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to …

Probiotic Therapy For Heart Failure: Investigating The Potential Anti-Hypertrophic Properties Of Probiotics, Grace L. Ettinger

Electronic Thesis and Dissertation Repository

Heart failure patients face a five-year 50% mortality rate, due to impaired cardiac function and hypertrophy of the heart. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Considering the established cardiovascular benefits of some probiotics, including reduction of cholesterol and hypertension, it was hypothesized that probiotics can improve outcomes of heart failure. Probiotics or controls were administered orally to an animal model for heart failure. Those receiving probiotics showed a significant improvement in cardiac hypertrophy and an attenuation of heart failure compared to control. No changes in gut microbial composition occurred. …

Sub-Inhibitory Antibiotics Enhance Virulence, Persistence, And Pathogenesis Of Uropathogens, Lee W. Goneau

Electronic Thesis and Dissertation Repository

In addition to their bactericidal effects, antibiotics are potent signal mediators at sub-inhibitory levels in the environment. The ability to modulate community structure in this niche raises concerns over their capacity to influence pathogenesis in patients during antibiotic therapy. This concept forms the basis of this thesis, and is explored using models of prophylactic therapy for recurrent urinary tract infection (UTI) management.

Sub-inhibitory ciprofloxacin, ampicillin, and gentamicin were found to augment virulence in vitro, increasing adherence and urothelial cell invasion in uropathogenic Escherichia coli (UPEC) and Staphylococcus saprophyticus. In addition, biofilm formation was increased, and swarming motility decreased. …

The Effect Of Nutrition On The Microbiome In Pregnant Women And The Use Of Micronutrient Supplemented Probiotic Yogurt To Improve Outcomes, Megan Kathleen Enos

Electronic Thesis and Dissertation Repository

The aims of this study were to characterize the gut, oral, and vaginal microbiotas of pregnant women with varying nutritional status and to determine if daily supplementation with probiotic yogurt containing Lactobacillus rhamnosus GR-1 and ground Moringa leaves, would ‘normalize’ the microbiota and improve pregnancy outcomes. For the latter, a clinical study was performed in Mwanza, Tanzania, in which consumption of yogurt for 88 ± 31 days, starting from the second trimester, did not result in excess weight gain, altered pregnancy outcomes or increased blood nutrient levels compared to an under-nourished group not taking the yogurt. Ingestion of the probiotic …

Characterization Of Staphylococcus Aureus Lipase, Vithooshan Vijayakumaran

Electronic Thesis and Dissertation Repository

USA300, a strain of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA), has become prevalent in the community. Colonization of human skin requires mechanisms that allow this bacterium to overcome the innate immune defenses on the skin, including secretion of antimicrobial lipids. Antimicrobial lipids inhibit S. aureus growth and induce the staphylococcal proteolytic cascade, producing aureolysin (Aur) which processes the lipase glycerol ester hydrolase (Geh). Nearly all S. aureus strains secrete Geh, yet little information exists concerning its function. Using purified Aur and Geh we confirm that aureolysin processes proGeh to Geh. We then confirmed that geh was required for lipase activity …

Role Of Branched-Chain Amino Acid Transporters In Staphylococcus Aureus Virulence, Sameha Omer

Electronic Thesis and Dissertation Repository

Branched-chain amino acids (BCAAs) act as effector molecules that signal a global transcriptional regulator, CodY, to regulate virulence factors in nutrient depleted environments. Staphylococcus aureus contains three putative BCAA transporters (BrnQ1, BrnQ2, BrnQ3) whose role in BCAA uptake is unknown. We hypothesize that BrnQ transporters are involved in BCAA uptake and contribute to virulence in S. aureus by modulating CodY activity. Results from radioactive uptake assays indicate that BrnQ1 is the predominant BrnQ transporter of isoleucine, valine and leucine. Meanwhile, BrnQ2 is more specific for isoleucine. Furthermore, only the lack of BrnQ1 hinders growth of S. aureus in chemically-defined media …

The Interaction Of The Human Adenovirus E1a Protein With The Human Dref Transcription Factor, Kris M. James

Electronic Thesis and Dissertation Repository

The human adenovirus (HAdV) E1A protein is the first protein produced post-HAdV infection, and serves two main functions. The first is to modulate host and viral transcription. The second is to induce host cell cycle progression to S phase, to promote an optimal environment for viral replication. E1A performs its functions by binding and manipulating over 50 cellular factors. Interestingly, I found that E1A is capable of interacting with the poorly characterized human DNA replication-related element-binding factor (hDREF). hDREF is a transcription factor associated with the expression of several genes related to the cell cycle. I hypothesized that the interaction …

Human Adenovirus E1a Binds And Retasks Cellular Hbre1, Blocking Interferon Signalling And Activating Virus Early Gene Transcription, Gregory J. Fonseca

Electronic Thesis and Dissertation Repository

Upon infection, human adenovirus (HAdV) must block interferon signaling and activate the expression of its early genes to reprogram the cellular environment to support virus replication. During the initial phase of infection, these processes are orchestrated by the first HAdV gene expressed during infection, early region 1A (E1A). E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. We have identified hBre1/RNF20 as a novel target of E1A. hBre1 is an E3 ubiquitin ligase which acts with the Ube2b E2 conjugase and accessory factors RNF40 and WAC1 to monoubiquitinate …

Staphylococcus Aureus Response To Long Chain Antimicrobial Fatty Acids, Benjamin Arsic

Electronic Thesis and Dissertation Repository

Staphylococcus aureus is a common pathogen that has the ability to infect virtually every tissue and organ system of the body. Despite its propensity to cause invasive infection, S. aureus is also a commensal organism, asymptomatically colonizing ~25% of the population. Much research has gone into resolving this paradox, focusing on both human and bacterial factors that may contribute to colonization. Antimicrobial fatty acids present on the skin and in the nasal mucosa are important components of the innate immune system, and thus we undertook to further understand how S. aureus responds to these fatty acids, and how this response …