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Mutual Regulation Of Crp And N(Epsilon)-Lysine Acetylation In Escherichia Coli, Robert James Davis

Dissertations

Post-translational modifications, such as N(epsilon)-lysine acetylation, are known to alter the behavior of transcriptional regulators in eukaryotes, but very little is known about the consequences of acetylation on transcriptional regulation in bacteria. Here, I provide evidence that a global transcriptional regulator of carbon metabolism, cAMP Receptor Protein (CRP), promotes both enzymatic and non-enzymatic lysine acetylation in E. coli. Non-enzymatic lysine acetylation occurs when cells ferment acetate, such as during growth on high concentrations of glucose. Intriguingly, CRP can be non-enzymatically acetylated on several lysines, including lysine 100 (K100). I provide evidence that neutralization of the K100 positive charge, as would …

Characterization Of Quorum-Quenching Lactonases And Plp-Dependent Aminotransferases: Structure, Mechanism And Alternative Turnover, Romila Nina Mascarenhas

Dissertations

Cell-to-cell communication by bacteria is essential for the regulation of gene expression important in colonization, biofilm formation, virulence and other processes. This communication is called "quorum-sensing" and is mediated by small molecules called autoinducers. One major class of autoinducers used by gram-negative bacteria is N-acyl homoserine lactones (AHL's). Enzymes capable of disrupting this communication are called "quorum-quenching" catalysts and have proven to be invaluable biochemical tools for understanding quorum-sensing pathways. Quorum-quenching enzymes hold promise for application in anti-biofouling, agriculture, aquaculture, bioremediation and other synthetic biology settings. However, the mechanisms that these enzymes use to recognize and process their substrates are …

The Role Of Notch-1-Mediated Repression Of Pten On Growth And Cancer Stem Cell Survival In Trastuzumab Resistant, Her2+ Breast Cancer, Andrew Thomas Baker

Dissertations

Trastuzumab targets the ErbB2 (HER2) receptor on breast cancer cells to attenuate HER2 driven tumor formation. Trastuzumab reduces both downstream PI3K/Akt and MAPK pathway signaling as well as the breast cancer stem cell (BCSC) population. BCSCs are hypothesized to be responsible for tumor recurrence, metastasis, as well as drug resistance. Today, resistance to trastuzumab remains a major clinical problem for women diagnosed with HER2+ breast cancer. Attenuation of PI3K/Akt and MAPK pathways may occur through the tumor suppressor, PTEN. Women with HER2+ breast tumors expressing less PTEN and increased PI3K/Akt or MAPK activity have worse overall outcome. Previously we have …

Role Of The Interleukin-22 And Stat3 Signaling Pathway In Gut Barrier Maintenance Following Intoxication And Burn Injury, Adam M. Hammer

Dissertations

More than half of patients admitted to burn centers for treatment have measurable alcohol levels in their blood. Intoxication that precedes a traumatic burn injury leads to worsened patient outcomes, including higher rates of infection and sepsis. The intestines harbor the largest concentration of bacteria in the human host. Any disruption of the intestinal barrier as a result of intoxication and burn injury could contribute to infection and/or sepsis from gut-derived microbes. Previous work has demonstrated that the intestinal barrier becomes leaky, and that administration of interleukin-22 (IL-22) significantly reduces gut barrier leakiness following alcohol and burn injury. However, the …

Characterization Of The Effect Of Aeration On A Commercially Available Fibrin Sealant For Use In Wound Therapy, Adam Robert Marek

Dissertations

Wound care affects millions of people worldwide each year, and the need for an effective wound therapy still exists. The aim of this study was to characterize a novel, aerated biopolymer, fibrin foam, which is generated through a patented mixing process using a commercially-available fibrin sealant. This research developed a distinct preparation of fibrin foam that creates a porous environment with improved wound healing properties. With this fibrin foam, characterization assessments were performed, including evaluation of mixing parameters, biocompatibility, and biomechanical strengths.

Fibrin foam is created by performing six passes through a mixing device, which generates a foam matrix with …

Frnk Regulatory Complex Formation With Fak Is Regulated By Erk Mediated Serine 217 Phosphorylation, Taylor J. Zak

Dissertations

Focal adhesion kinase related non-kinase (FRNK) is an endogenous inhibitor of focal adhesion kinase (FAK) that has traditionally been used to inhibit FAK signaling in a variety of experiments and is also an important endogenous regulator of FAK signaling. More recently, FRNK has been shown to be of increasing importance in some pathologic conditions. Despite the increasing importance of FRNK, the molecular mechanism by which it functions remains unclear. In addition, FRNK contains several phosphorylation sites with unknown importance and function. Here I hypothesize that FRNK can inhibit FAK by binding directly to FAK within focal adhesions. Furthermore, I propose …

The Effects Of Alcohol And Traumatic Brain Injury On Neural Stem Cell Responses, Son Trung Ton

Dissertations

Traumatic brain injury (TBI) is a major source of disability in modern societies. However, there are no good pharmacological strategies for treating long-term TBI complications because we do not fully understand the injury processes that occur afterward. Moreover, a significant percent of patients entering the emergency room with TBI have a blood alcohol level above the legal limit. Alcohol use and intoxication is a widespread issue in our society. Binge drinking is the most common way in which alcohol is consumed, and alarmingly, Americans of all age groups binge drink on a frequent basis. Despite the prevalence of alcohol intoxication …

Endocytic Vesicle Rupture In The Pathogenesis And Propagation Of Neurodegenerative Proteinopathies, William P. Flavin

Dissertations

Numerous pathological amyloid proteins spread from cell-to-cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is therefore key to understanding the progressive nature of such neurodegenerative proteinopathies. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to induce the rupture of intracellular vesicles following endocytosis, as well as to elucidate the cellular consequences of this damaging mechanism of invasion. We observed that the ability to induce vesicle rupture is a conserved feature of …

Aquatic Insect Community Structure And Secondary Production In Coastal Wetland Ponds Of The Copper River Delta, Alaska: Influence Of Water Temperature And Macrophyte Community Structure, Chantel Caldwell

Master's Theses

The Copper River Delta (CRD), southcentral Alaska, is one of the world’s largest continuous coastal wetlands and is largely composed of sloughs, lakes, and ponds. Due to coastal topography the east side of the Copper River (East Delta) is disproportionately impacted by a cold continental air mass. Wetland communities of the CRD were also impacted by a 9.2 magnitude earthquake in 1964 that shifted the more coastal portion of the CRD from tidally influenced ponds to freshwater ponds.

The West-East temperature gradient across the CRD coupled with landscape type (uplifted marsh (UM) and outwash plain (OP)) creates four regions (West-UM, …

Population Genetics And Distribution Of The Oriental Weatherfish, Misgurnus Anguillicaudatus, In Chicago Area Waterways, John Belcik

Master's Theses

Oriental Weatherfish (Misgurnus anguillicaudatus), native to Southeast Asia, are freshwater fish belonging to the family Cobitidae (loaches). They are benthic fish with the ability to breathe atmospheric air, and exhibit an unusual behavior of swimming vertically in the water column in response to changes in barometric pressure. Oriental Weatherfish appear to be successful invaders to North American waterways. Within the last century they have been sighted in freshwater systems throughout the United States, yet the distribution and source of this invasion are largely unknown. This study investigates the distribution patterns, surveying methods, and population genetics of the Oriental Weatherfish within …

The Role Of Phage Trnas In The Evolution Of Codon Usage Biases In Giant Pseudomonas Phage Phikz And El, Stephanie Steidel

Master's Theses

As the most abundant and diverse biological agents in the biosphere phage have significant roles in microbial ecology, acting both as lethal bacterial parasites and vehicles of horizontal gene transfer. Phage/host coevolution drives optimization of phage codon usage for use of host translational machinery, thus lowered correspondence between phage and host codon usage reduces viral fitness. Some phage may partially bypass host translational selection on their codon usage by encoding their own tRNAs, although the effects of these tRNAs on phage codon usage and translation has not been well examined. This work explores the influence of phage encoded tRNAs on …

Utilizing Single-Molecule Fret Methods To Study Conformational Changes In Trim5Α, Margret Suzanne Bradley

Master's Theses

Single-molecule FRET (smFRET) is a method by which dynamic conformational changes can be monitored in a protein microscopically and in real time. smFRET relies on the creation of FRET (Förster Resonance Energy Transfer) between small molecule fluorophores conjugated to the biomolecules of interest. FRET efficiency allows calculation of interfluorophore distances. Changes in FRET efficiency represent changes in protein conformation which can inform further structural and molecular studies of the protein of interest. For example, in the Campbell Lab, we study the protein TRIM5α, an antiretroviral cellular protein which can cause premature dissociation of the HIV capsid core by an unknown …

The Effects Of Treating Multispecies Biofilms With Bacteriophage, Alexandria Francis Cooper

Master's Theses

Biofilms can be found in any environment within proximity to water and are problematic in an assortment of industries. Numerous efforts have been employed to dislodge biofilms including bacteriophages (viruses that infect bacteria). Phage remediation is a promising solution for combatting biofilms that form on catheters in long term use patients. These biofilms often result in catheter-associated urinary tract infections (CAUTIs) and are the most common type of healthcare related infection reported. Additionally, they result in longer hospital stays and increased morbidity. To date, most of the research on the topic focuses on single species biofilms, despite their rarity in …

Nutrient Removal And Vegetation Recovery Through Successive Harvesting Of The Invasive Hybrid Cattail (Typha X Glauca) In Great Lakes Coastal Wetlands, Kelsey Berke

Master's Theses

Excess nutrient levels have led to invasive hybrid cattail (Typha × glauca) dominance in many Laurentian Great Lakes coastal wetlands. Its ability to alter wetland structure and function has made T. × glauca a target invasive species with respect to Great Lakes coastal wetland

restoration. This study investigated the effect of harvesting invasive T. × glauca biomass on nutrient removal, specifically nitrogen and phosphorus, and plant diversity recovery in Great Lakes coastal wetland ecosystems. This study demonstrated that without considering external nutrient inputs and outputs, a single harvest at Cheboygan Marsh, MI could remove ~2% of the total N and …

Utilizing Genetic Techniques To Identify Amino Acids Within The Putative Glycosyltransferase Sypq That Are Essential For Its Role In Biofilm Formation By Vibrio Fischeri, Mary Kathryn Flaherty

Master's Theses

The polysaccharide component of the V. fischeri biofilm is produced by proteins encoded by the symbiosis polysaccharide (syp) locus. The syp locus encodes 18 genes, six of which are putative glycosyltransferases (GTs). One of these GTs, sypQ, encodes a putative family 2 glycosyltransferase (GT-2). The mechanism by which SypQ facilitates biofilm formation is not yet understood. To uncover which specific residues are essential for the function of SypQ, I utilized Bioinformatics, site-directed mutagenesis, and random mutagenesis. The Bioinformatics identified putative GT-2 motifs in the SypQ primary sequence. The mutagenesis (both site-directed and random), identified five residues (D95, D151, S153, D236, …

The Novel Characterization Of Extracellular Vesicles Containing Proteins Which Have Been Implicated In The Pathogenesis Of Hiv Associated Neurocognitive Disorders, Grace Ispas

Master's Theses

Although Anti-Retroviral Therapy (ART) is practiced, HIV-1 positive individuals still experience HIV-Associated Neurocognitive Disorders (HAND), collectively described as the presentation of neurocognitive, behavioral and motor dysfunctions that decrease the quality of life, while increasing the mortality in ART suppressed HIV-1 positive patients.

Current literature suggests that extracellular vesicles are involved in the pathogenesis of HAND as they are believed to be transferring HIV-1 proteins to nearby neuronal cells. Although most studies assume homogeneity among populations, characterizing cellular proteins or RNA levels in bulk, we hypothesize that distinct populations of extracellular vesicles are released and that environmental conditions, including viral infections, …

Potential Efficacy Of Targeting Mll1 In Breast Cancer, Austin Gable Holmes

Master's Theses

In 2017, it is estimated that breast cancer will be the most prevalent newly diagnosed cancer in females, at 30% in the U.S. alone1. From the early 1990s to 2014 the death rate in females from breast cancer has dropped by 10% but still stands as the second highest cancer related death in females in the U.S.2. One of the biggest hurdles in breast cancer research is disease heterogeneity. New mechanisms of disease development and progression are encountered frequently. One mechanism studied in the past decades is epigenetics. It has been theorized that the cancer epigenome can maintain an abnormal …

Phenotyping Temperature-Sensitive Coronaviruses, Amani Eddins

Master's Theses

Coronaviruses (CoVs) can cause a range of symptoms; from a mild common cold to life threatening acute respiratory distress syndrome (ARDS) upon infection. These emerging viruses have the ability to be highly pathogenic and detrimental to the human population. Two prime examples of CoV emergence, SARS-CoV (Severe Acute Respiratory Syndrome) and MERS-CoV, (Middle Eastern Respiratory Syndrome), which exhibits the pandemic potential of emerging CoVs that gain human tropism. Given the increasing potential of an emerging CoV outbreak there is a state of urgency to develop vaccines that will help protect the human population against current and future circulating strains. The …

Optimizing Clean Catch Urine Collection And Its Applications In Urinary Microbiome Studies, Danielle Johansen

Master's Theses

Clean catch was developed before the advancement of more sensitive assays for culturing urinary bacteria. I reassessed clean catch, endeavoring to optimize this method. Periurethral swabs and voided urines were routinely collected, while analyzing different aspects of the method. I determined that midstream urine contained the least amount of vulvo-vaginal contamination and that the time of collection did not impact the results. I also determined that the use of antiseptic wipes prior to void increased the level of vulvo-vaginal contamination. A small cohort of females collected daily urines over a 17-day period and kept a strict alcohol diary. Females who …

Dexamethasone Treatment Effects On H3k27me3 Chromatin Organization Is Related To Nk Cell Immune Dysregulation, Michael Sarafeno Misale

Master's Theses

It is well-established that psychological stress reduces natural killer (NK) cell immune function. This reduction is mediated by stress-induced release of glucocorticoids (GC), which can suppress immune function. Associated with suppression of a particular immune function are GC induced histone-epigenetic marks. Histone-epigenetic marks are responsible for the organization and compartmentalization of genomes into transcriptionally active euchromatin domains that are localized to the interior of the nucleus. Transcriptionally silent heterochromatic domains are enriched with methylated epigenetic marks and are localized to the nuclear periphery. The purpose of this investigation was to assess the influence of GC on H3K27me3 chromatin organization by …

Defining The Roles Of The Lipoic Acid Ligases In Promoting Staphylococcus Aureus Metabolic Homeostasis And Virulence, Irina Laczkovich

Master's Theses

Staphylococcus aureus is a major human pathogen known to cause disease in a wide range of tissues. In order to thrive in such diverse environments, S. aureus uses multiple adaptive traits such as trace metal/nutrient acquisition, shifts in metabolic activity, and expression of detoxification systems, all of which allow the bacterium to proliferate and survive in nutritionally deficient and inhospitable environments.

One essential metabolite used by S. aureus is lipoic acid, a cofactor of enzyme complexes used in aerobic metabolism, fatty acid biosynthesis, glycine detoxification, and maintenance of redox homeostasis. Prior studies in the lab used a genetic approach to …

Functional Studies Of Novel Mosquito Stage-Specific Genes In The Malaria Parasite, Plasmodium Berghei., Kaitlyn Kiernan

Master's Theses

The development and survival of the malaria parasite Plasmodium within its mosquito vector is not very well understood. When the parasite is taken up by an Anopheles mosquito, it is exposed to digestive enzymes, reactive oxygen species, increasing pH, decreasing temperature, and a diverse microbial flora. Our lab is studying the adaptive mechanisms underlying parasite survival in this hostile environment, one of which is movement out of the midgut. In order to escape the midgut, the parasite develops into an elongated, motile form called the ookinete. I am investigating two genes, thioredoxin-like protein 1 (trxl-1) and subpellicular microtubule protein 1 …

Multifunctional Regulatory T Cells From Human Umbilical Cord Blood And The Role Of Tumor Necrosis Factor In Immune Homeostasis, Alexander Nelson

Master's Theses

Regulatory T cells (Tregs) are required to suppress inflammation and prevent autoimmunity. During fetal development Tregs are crucial to maintain tolerance between mother and child. After birth, neonates require tolerance to avoid harmful immune responses to foreign antigens in food and allow colonization with commensal microbes. We demonstrate a propensity for T cells in human umbilical cord blood to differentiate into Tregs in response to antigen receptor stimulation ex vivo. Cord blood-derived Tregs potently suppress T cell proliferation, but also produce pro-inflammatory cytokines known to activate innate immune responses. These results suggest that antigen exposure during early life results in …

Chronic Effects Of Lead Exposure On Atherinops Affinis (Topsmelt): Influence Of Salinity And Organism Age, Erik Joseph Reynolds

Master's Theses

Pb (lead) appears in the environment as a consequence of both natural and anthropogenic processes. Mining, smelting, coal burning, lead acid batteries, and cement manufacturing substantially release Pb into aquatic environments. The goal of this study was to determine the influence of salinity and organism age on chronic toxicity of Pb to Atherinops affinis (topsmelt) in support of development of a species sensitivity distribution (SSD). Species sensitivity distributions assist in ecological risk assessments and establishing quality criteria for contaminants. Three chronic exposure studies were conducted for 28 days in a water flow-through testing system. Survival, standard length, dry weight, and …

Characterization Of Inhibitory Molecules Produced By Lactobacillus Crispatus, Giuseppe Anthony Pistone

Master's Theses

Lactobacillus species are widely accepted as beneficial bacteria of the human microbiota1-8. Lactobacilli spp. are well documented to inhibit pathogens by production and secretion of hydrogen peroxide (H2O2), organic acids, and/or proteinaceous bacteriocins into their environment. Most research attributes bactericidal activity of cell free supernatant (CFS) to H2O2 and/or lactic acid. Here, I demonstrate that CFS from a clinical isolate of Lactobacillus crispatus contains a molecule(s) that inhibits uropathogenic Escherichia coli (UPEC) colony formation independently of H2O2 and organic acids. Physiologic concentrations of H2O2 and organic acids produced by L. crispatus do not inhibit UPEC colony formation. Incubation of UPEC …

Cellular Determinants Of Coronavirus Entry Routes, James Thomas Earnest

Dissertations

Coronaviruses (CoVs) represent some of the greatest modern threats to global health. CoVs are zoonotic viruses that generally cause respiratory or enteric infections. The ability of CoVs to move between species and into human populations ensures that CoVs will remain important and dangerous pathogens. Therefore, understanding how CoVs infect different hosts is vital to human health.

CoVs are enveloped and must undergo fusion of viral and host membranes to initiate infection. Viral glycoproteins, called Spike (S) proteins, are responsible for host cell binding and carrying out the membrane fusion reaction. S proteins store energy in their folded, pre-fusion conformation that …

Behavioral Symptoms, Functional And Cognitive Recovery Post-Mild Traumatic Brain Injury, Seema Nasser

Dissertations

Mild traumatic brain injury (MTBI) accounts for 80–90% of the nearly two million traumatic brain injuries (TBI) that occur each year. The psychological consequences of MTBI can be extensive and can persist well beyond the acute injury, profoundly impacting the lives of the individual, their families, and society. A substantial number of MTBI patients suffer depressive mood, fatigue, and poor sleep (i.e., behavioral symptoms) for weeks and months post-injury. These symptoms reduce quality of life and delay the return to previous cognitive and functional status. Behavioral symptoms of depressed mood, fatigue, and poor sleep commonly co-occur and thus may constitute …

Examining The Regulation Of Kv7 K+ Channels In Airway Smooth Muscle Cells And Their Potential As Novel Therapeutic Targets For The Treatment Of Asthma, Jennifer Haick

Dissertations

Asthma is a disease characterized by nonspecific and exaggerated airway narrowing, termed airway hyperresponsiveness (AHR), which involves the excessive contraction of airway smooth muscle. Despite the fact that airway smooth muscle is widely studied and understood to play a role in AHR, little is known about the specifics of that role. Our laboratory recently found that Kv7 potassium (K+) channels are expressed in airway smooth muscle cells (ASMCs). Kv7 channels are voltage sensitive K+ channels whose outward flux of K+ ions promotes a negative resting membrane voltage in excitable cells, thereby opposing electrical excitability. Inhibition of K+ channels is known …

Characterization Of Multi-Albumin Pegylated Complexes Synthesized Using "Click" Chemistry As Drug Delivery Systems, Jonathan Alejandro Hill

Dissertations

Due to its ability to transport hydrophobic materials through the bloodstream, human serum albumin has become an intriguing component for drug delivery systems. The most pressing concerns with current albumin-based systems are the risk of pathogenic transfer associated with human-sourced blood proteins, the negative effect on osmotic pressure due to administration of a large dose of albumins, the potential immune response and loss of biological activity due to heavy modification of these albumins, and the need for organic solvents for preparation of these products. Key residues in the two main drug binding sites, DS1 and DS2, are conserved in bovine …

The Female Urogynecological Microbiome: Determining Niche Specificity, Krystal Thomas-White

Dissertations

Contrary to dogma, urine is not sterile. Recent discoveries show that the female urinary bladder contains a unique microbiome,with differences in healthy and disease states. This female urinary microbiome (FUM) is distinguishable from vulvo-vaginal contamination; yet, only the few centimeters of the urethra separate these two niches. Recent studies show that the most common bladder organisms are Lactobacillus, Gardnerella, Streptococcus, Corynebacterium, and a diverse group of anaerobes, the same genera commonly found in the vagina. Thus, we asked if the bladder and vaginal microbiomes are interconnected.

Previous work suggests a link between these two niches. The vaginal tract of reproductive …