Master of Engineering, Case Western Reserve University, 2022, Biomedical Engineering

Mucin-type O-glycosylation is an abundant yet understudied post-translational modification of proteins in metazoans, initiated by the polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) of which twenty are known in humans. O-glycosylation is involved in many biological processes including intracellular signaling and cell-cell interactions. GalNAc-T mutations contribute to several disease states such as cancer. Previous characterization of the GalNAc-Ts has revealed unique preferences for peptide sequence and prior substrate glycosylation. Herein, another paradigm by which the GalNAc-Ts select peptide targets is investigated: flanking substrate charge. Twelve GalNAc-Ts have been characterized against substrates containing different N-/C-terminal charges, demonstrating unique and overlapping charge preferences. Electrostatic models, elevated ionic strength, and molecular dynamics simulations reveal that GalNAc-Ts selectivity is indeed modulated via charge-charge interactions. Michaelis-Menten kinetics revealed significant variation in kinetic parameters, particularly Vmax. Overall, this work reveals that charge-charge interactions are another important but previously overlooked factor that uniquely modulates GalNAc-T specificity.

Committee: Thomas Gerken (Advisor); Mei Zhang (Advisor); Xinning Wang (Committee Member); Ryan Arvidson (Committee Member); Sam Senyo (Committee Chair) Subjects: Biochemistry; Biomedical Engineering

Doctor of Philosophy, The Ohio State University, 2021, Biochemistry Program, Ohio State

Cell adhesion is an incredibly important process for multicellular life and is essential for development and maintenance of tissues, immunological response, cancer suppression and mechanosensation. The cadherin superfamily of calcium-dependent cell-adhesion proteins is a large group of diverse glycoproteins which play roles in the processes listed above. The superfamily can be broken down in roughly three subfamilies: classical cadherins, clustered protocadherins, and non-clustered protocadherins. Of those subfamilies, the cadherins in the non-clustered protocadherins have the most diverse range of adhesive functions. To mediate adhesion, cadherins make use of their extracellular domain, which is composed of tandem extracellular cadherin (EC) repeats. Within the repeat sequence, conserved motifs are present encoding for residues which bind three calcium ions at the linker region between repeats, thus imparting rigidity to the extracellular domain. Calcium binding is essential for adhesive function to occur. Here, I focus on several unique non-clustered protocadherins which use their ectodomains to help support the development and tissue morphogenesis of various systems. My work on the 7D-cadherins has focused primarily on cadherin 17 (CDH17) and its homophilic adhesion relevant for the function of the intestinal epithelia. In addition, its sibling, cadherin 16 (CDH16), is expected to mediate similar adhesion in the kidney epithelia. Structures of CDH17 EC1-2 reveal that the 7D-cadherins are unique and distinct from their relatives, the classical cadherins, in that they lack the tryptophan necessary for the conserved strand-swap interaction observed in classical cadherins. Bead aggregation assays using the full ectodomain of CDH17 as well as N- and C-terminal truncation series reveal that CDH17 relies on its EC7, but not its EC1 repeat, to carry out trans adhesion. A mutation in EC1 interferes with aggregation of the full-length ectodomain, indicating the entire e (open full item for complete abstract)

Committee: Marcos Sotomayor (Advisor); Richard Swenson (Committee Member); Ross Dalbey (Committee Member); Kotaro Nakanishi (Committee Member) Subjects: Biochemistry; Biology; Biophysics

Doctor of Philosophy, Case Western Reserve University, 2020, Biochemistry

Mucin type O-glycosylation is one of the most common and diverse protein modifications in humans. The process is initiated by a large family of N-acetylgalatosamine transferases (GalNAc-T1 to 20), that transfer the GalNAc sugar onto specific serine (Ser) and threonine (Thr) acceptor residues on target proteins. The underlying molecular mechanisms that govern acceptor Thr/Ser site selection by the GalNAc-T are not well understood. Mutations and abnormal expression of individual GalNAc-T isoenzymes lead to alterations in site-specific O-glycosylation, which have been associated with many cancers and metabolic syndromes. Previous substrate specificity studies have shown that the peptide sequence and the prior O-GalNAc glycosylation on a substrate dictate the acceptor site selection by GalNAc-Ts. Structurally, nearly all GalNAc-Ts contain a catalytic domain and a lectin domain connected by a flexible linker. The enzyme activity is enhanced by the lectin domain recognizing remote prior glycosylation (>5 residues away from the acceptor) in an N-or C-terminal direction, and a small number of transferases show rate enhancement by the catalytic domain recognizing neighboring prior glycosylation (<5 residues away). In this thesis we have begun to show how the acceptor site preferences of GalNAc-T isoenzymes are fine-tuned by a combination of multiple enzyme-substrate interactions, guided by enzyme domain specificities that ultimately results in a defined pattern of O-glycosylation. In chapter 1, we show that GalNAc-Ts have intrinsic preference for acceptor Thr residues over Ser residues independent of the flanking peptide sequences. Interestingly the Thr/Ser rate ratio varies between GalNAc-T subfamilies thus adding to the increasing dimension of GalNAc-T substrate specificity. In chapter 2, we show that GalNAc-T2's preference for both N- and C-terminal remote prior glycosylation is primarily based on its short flexible linker that provides rotational and translationa (open full item for complete abstract)

Committee: Thomas Gerken (Advisor); Martin Snider (Committee Chair); William Merrick (Committee Co-Chair); Vincent Monnier (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Miami University, 2016, Microbiology

Acinetobacter baumannii is an opportunistic nosocomial pathogen that has a global impact on human health. The capacity of A. baumannii to cause a wide variety of severe infections, combined with the emergence of multi-drug resistant isolates, present significant challenges toward the development of new therapies to treat A. baumannii infections. Biofilm formation on abiotic surfaces, which is a key factor in the persistence of this pathogen, is controlled by the BfmRS two-component regulatory system (TCS). However, the role of this TCS in other Acinetobacter isolates is not understood. The work presented herein demonstrates that the BfmR response regulator provides conserved global regulatory functions, which includes the regulation of adherence and pathogenicity factors, in both A. baumannii and Acinetobacter nosocomialis. In contrast to the interaction of A. baumannii with abiotic surfaces, the mechanism by which this pathogen regulates attachment and virulence factors in biotic environments is unknown. Our work provides insight into this pathway by demonstrating that A. baumannii responds to mucin, a major component secreted by lung epithelial cells particularly during respiratory infections, which triggers a cascade of responses that allow this pathogen to acquire critical nutrients and enhance the expression of virulence factors in the host environment. Furthermore, along with data collected by others, our findings shed light on the important role of mucin in the effectiveness of antibiotics toward A. baumannii in biotic environments where this glycoprotein is present. Taken together, these findings provide a deeper understanding of how A. baumannii uses a complex regulation of adherence and virulence factors in response to specific environmental signals to promote its persistence and pathogenicity within abiotic and biotic environments.

Committee: Luis Actis PhD (Advisor); Mitchell Balish PhD (Committee Member); Rachael Morgan-Kiss PhD (Committee Member); Joseph Carlin PhD (Committee Member); Nicholas Money PhD (Committee Member) Subjects: Genetics; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2016, Chemistry

Many proteins of eukaryotic cells are known to be O-glycosylated. Glycoproteins with heavily O-glycosylated mucin domains provide important biological functions in a cell: i.e., protection from pathogens, cell-to-cell adhesion and intracellular protein trafficking. Mucin-type O-glycosylation occurs in the Golgi complex and begins with the transfer of GalNAc from UDP-GalNAc onto Ser/Thr residues of polypeptides. This step is catalyzed by a large family (20) called N-a-acetylgalactosaminyl transferases (ppGalNAc-T's) and forms the GalNAc-a-O-Ser/Thr product. Subsequent elongation is performed by specific glycosyltransferases, producing a variety of glycans. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both subfamilies possess variable activities against glycopeptide substrates. Structurally, 19 isoforms contain a C-terminal catalytic domain linked via a flexible linker to an N-terminal ricin-like lectin domain. The (glyco)peptide substrate specificities of the ppGalNAc-T transferases and the roles of the catalytic and lectin domains in glycopeptide glycosylation still remain largely unknown. Based on the systematic random peptide approach created by the Gerken Lab, I have determined the glycopeptide substrate specificities of several ppGalNAc-T isoforms. A series of (glyco)peptides were created in order to specifically probe the functions of the catalytic and lectin domains in terms of neighboring (1-5 residues) and remote prior glycosylation (6-17 residues) from an acceptor site, respectfully. Using several glycopeptide-preferring isoforms, glycosylation was observed from -4, -3, -1 and +1 relative to a neighboring GalNAc-O-Thr, which I attributed to specific GalNAc-O-Thr binding at the catalytic domain. The other series of glycopeptides contained a GalNAc-O-Thr near the C- or N- terminus of the substrate to address the directionality preferences of the lectin domain. Results with several peptide- and glycopepti (open full item for complete abstract)

Committee: Thomas Gerken (Advisor); Irene Lee (Committee Chair); Michael Zagorski (Committee Member); Paul Carey (Committee Member); Michael Harris (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, University of Akron, 2015, Integrated Bioscience

Understanding microbial treatment strategies represents a unique challenge for integrative biologists. From one viewpoint, the metabolic diversity of naturally occurring bacteria could be utilized to develop passive treatment strategies that target specific systems impacted by environmental contamination. On the other hand, from a medical perspective many bacteria are capable of establishing residence within host environments often resulting in difficult to treat infections. The purpose of this research was to explore two specific scenarios, acid mine drainage (AMD) and cystic fibrosis (CF), as a means to further develop potentially useful microbial treatment strategies. In order to develop effective passive treatment options for water pollution caused by AMD, it becomes necessary to first evaluate how natural soil bacteria respond to sustained AMD intrusion. Data presented shows how the population of soil microorganisms from an AMD site exhibiting “sheet flow” characteristics shifts from a highly diverse community towards a less metabolically robust community composed of higher relative percentages of organisms capable of metabolic iron(II) oxidation. Data is also shown for depth dependent organization of microbial communities through a “mature” iron mound that has formed as a result of continuous iron(II) oxidation and subsequent precipitation of various iron(III)-(hydr)oxides. From a medical perspective, more clinically relevant microbial treatment methodologies are necessary to combat infections such as those commonly caused by Pseudomonas aeruginosa in the lungs of CF patients. Utilizing conditions that better approximate the chemical composition and bacterial population of the CF lung environment, various antimicrobial compounds (including two novel silver carbene complexes) were examined for efficacy against planktonic and biofilm phenotypes of multiple strains of P. aeruginosa. Data shows that mucin (a key component of the CF lung environment) concentr (open full item for complete abstract)

Committee: John Senko Dr. (Advisor); Donald Ott Dr. (Committee Member); Wiley Youngs Dr. (Committee Member); Robert Joel Duff Dr. (Committee Member); Rolando Juan Jose Ramirez Dr. (Committee Member) Subjects: Biology

Master of Science, The Ohio State University, 2014, Animal Sciences

Ohio has a 158 million dollar turkey industry. Gut growth and development occurs first before the turkey can realize its full muscle producing potential. Alternative and adjunctive approaches to decreasing the use of feed grade antibiotics are becoming an important area of research due to increasing consumer and legislative concerns with antibiotic resistance. Probiotics or supplemental dietary commensal microbes is one such potential approach as they can colonize the intestine, particularly in young animals with a relatively naive intestine microbiome. Intestinal mucosa is made up of mucin glycoproteins that play a key role in preventing the attachment and colonization of pathogenic bacteria. These proteins are made up of a protein backbone that is coded for by the MUC gene family. At hatch, the turkey intestine is relatively aseptic and therefore vulnerable to bacterial colonization from both commensal and pathogenic microbes. In this study, we determined the expression of MUC2, which codes for a secretory gel-forming mucin that is predominantly found in the small and large intestine, from immediately post-hatch through day 11 of age in poults fed a conventional starter diet, the starter diet supplemented with two commercial probiotics (A and B), or the starter diet supplemented with a commercial prebiotic. This was done by comparing the MUC2 transcription levels to the transcription level of a housekeeping gene. Multiple potential housekeeping genes were investigated and one, RPS13, was found to be stably expressed across all ages and treatments in the turkey poult. The effects of the supplemented diets on intestinal development were also analyzed. While MUC2 transcription increased with age, there were no significant effects due to diet. The intestinal parameters of villus height, area and crypt depth were all increased with supplementation of probiotic B and the prebiotic.

Committee: Michael Lilburn (Advisor); Macdonald Wick (Committee Member); Joseph Ottobre (Committee Member) Subjects: Animal Sciences

PhD, University of Cincinnati, 2014, Medicine: Epidemiology (Environmental Health)

Background: Small studies have found a connection between intestinal microbial colonization and risk of late-onset sepsis, but leave open questions about the generalizability of these findings. Also largely unstudied is the role of the host genotype in shaping the intestinal microbiome of the premature infant. Methods: The Novel Biomarkers Cohort is a large, prospective cohort of premature infants (born =32 weeks gestational age) enrolled from Neonatal Intensive Care Units (NICUs) in Birmingham, Alabama and Cincinnati, Ohio. Birmingham infants were compared Cincinnati infants to determine the degree to which findings regarding the microbiota may be generalizable between NICUs. Then, a nested case-control study of 66 infants was conducted to determine colonization patterns associated with late-onset sepsis. Finally, the microbiota of Cincinnati infants were analyzed to explore the contribution of host genetics to shaping the intestinal microbial community. Results: Of the NICUs, Birmingham preterm infants tend to have more stable bacterial communities during the first three weeks of life, with more Proteobacteria and Bacteriodes, and less Firmicutes than Cincinnati preterm infants. The composition of infant samples varied over calendar months and between postnatal weeks of life. Streptococcaceae and Clostridia were associated with sepsis in Birmingham and the Bacilli and the lack of Actinobacteria were associated with sepsis in Cincinnati. SNPs in specific fucosyltransferase and mucin genes were associated with higher or lower relative abundance of specific bacteria. However, these differences were not relevant to the patterns observed prior to sepsis.

Committee: Ardythe Luxion Morrow Ph.D. (Committee Chair); Christina Valentine Ph.D. (Committee Member); Ranjan Deka Ph.D. (Committee Member); John Harley M.D. Ph.D. (Committee Member); Marepalli Rao Ph.D. (Committee Member) Subjects: Epidemiology

PhD, University of Cincinnati, 2010, Medicine : Molecular and Developmental Biology

The respiratory epithelial cells lining the conducting airways play critical roles in mediating innate and acquired immune responses by participating mucociliary clearance, secreting anti-bacterial peptides, and release cytokines and chemokines to interact with immune cells. The forkhead box transcription factor, Foxa2, is normally expressed in respiratory epithelial cells lining conducting airways and in alveolar type II cells. Foxa2 is required for normal lung maturation, surfactant protein and lipid synthesis during lung development. However, the role of Foxa2 in regulating crosstalk between epithelium and immune system during lung development was unknown. In the present thesis, selective deletion of Foxa2 allele in the respiratory epithelium mediated by human surfactant protein C (SFTPC) promoter driven Cre recombinase during embryonic stage (E6.5-E12.5) was found to cause asthma-like phenotype including eosinophilic inflammation and goblet cell metaplasia in the neonatal mice. Loss of Foxa2 induced the recruitment and activation of myeloid dendritic cells (mDCs) and T helper 2 (Th2) cells in the lung, resulting in increased production of Th2 cytokines and chemokines, including interleukin 4 (IL-4), IL-13, IL-5 and thymus and activation-regulated chemokine (Tarc), and induced expression of goblet cell transcription factor Spdef. Expression of Foxa2 in the non-ciliated secretory epithelial cells (Clara cells) inhibited Spdef expression and goblet cell differentiation after allergen exposure, suggesting that Foxa2 and Spdef interacted within a genetic network that was associated with goblet cell differentiation. Spdef, SAM pointed domain Ets-like factor, normally expressed at low level in tracheal and bronchial epithelium, is significantly induced in goblet cells after IL-13 or allergen exposure at a Stat6 dependent manner. Expression of Spdef in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation in vivo. Spdef (open full item for complete abstract)

Committee: Jeffrey Whitsett MD (Committee Chair); James Wells PhD (Committee Member); George Leikauf PhD (Committee Member); Gurjit Hershey MD, PhD (Committee Member); John Shannon PhD (Committee Member) Subjects: Immunology