Life Sciences Commons^™

Role Of Micrornas In Intestinal Inflammation And Barrier Homeostasis After Alcohol And Burn Injury, Caroline J. Herrnreiter

Dissertations

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression. Within the intestinal epithelium, miRNAs play a critical role in gut homeostasis and aberrant miRNA expression has been implicated in various disorders associated with intestinal inflammation and barrier disruption. In this study, we sought to profile changes in intestinal epithelial cell miRNA expression after alcohol and burn injury and elucidate their impact on inflammation and barrier integrity. In a more targeted approach, we began by focusing on anti-inflammatory miRNAs that, when downregulated, could exacerbate inflammation and result in intestinal barrier disruption. Using a mouse model of acute ethanol intoxication …

Characterizing The Dynamic Localization Of Cmi In Early Drosophila Development, Asra Habibullah

Master's Theses

The COMPASS-like family of lysine methyltransferases, MLR/MLX complexes, are epigenetic regulators that are essential for normal development through the methylation of the fourth lysine residue on histone 3 (H3K4), a universal epigenetic mark associated with active transcription. This family of complexes is highly conserved from yeast to mammals and the genes encoding the human MLR complexes have been associated with various developmental diseases and cancers (Dingwall and Fagan, 2019). In D. melanogaster, the enzymatic methyltransferase core of this complex is composed of two proteins: Cara Mitad (Cmi, also known as Lpt) and Trithorax-related (Trr). Although these proteins have been shown …

Characterization Of The Full-Length Bag3 Protein And Stress Induced Formation Of Bag3-Z, Ahmed Gamal Abdalla Zied

Master's Theses

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone that we havepreviously shown localizes to the cardiac sarcomere and is critical for proteostasis and maintenance of normal sarcomeric function. Indeed, BAG3 loss in heart failure (HF) results in accumulation of ubiquitinated sarcomeric proteins, and depressed maximum force generating capacity (Fmax). However, how BAG3 is regulated in the cell is not well understood, with uncertainty about its structure and proteoforms. During our analysis of human heart tissue, BAG3 appears as a “doublet”, with one band at 74 kDa (BAG3-Z) and a second at a higher 85 kDa (BAG3-FL). Previous studies hypothesized the full-length …

Dynamic Control Of The Cardiac Calcium Pump, Sean Robert Cleary

Dissertations

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ into the endoplasmic reticulum of cells to establish a reservoir for Ca2+ signaling. In the heart, the activity of this transporter is tightly controlled via direct interactions with two competing regulatory micropeptides: phospholamban (PLB) and dwarf open reading frame (DWORF). PLB inhibits SERCA, while DWORF activates SERCA. These competing interactions determine cardiac performance by modulating the Ca2+ signals that drive the contraction/relaxation cycle. Previous studies indicated these SERCA-micropeptide interactions are Ca2+-sensitive; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. Here, FRET-microscopy demonstrated that this …

The Impacts Of Framework Mutations On Steady-State Kinetics Of An Aldolase Abzyme, Charles Andrew Mettler

Master's Theses

During affinity maturation, antibodies acquire point mutations that lead to enhanced binding strength to a particular ligand while simultaneously becoming more conformationally restricted as they become increasingly complementary to their ligand’s electrostatic geometry. This property has catalytic potential in transition-state (TS) stabilization whereby kinetic barriers (i.e., the energetic gap between the substrate and its TS) are reduced and reaction rates are enhanced. This basic mechanism is thought to underlie the majority of the rate enhancement observed for enzymes. A key integration of antibody binding specificity and strength with enzymatic catalytic strategy is the development of catalytic antibodies (“abzymes”). Abzymes are …

Structural And Transient Kinetic Analysis On Mechanism-Based Inactivators Of Human Ornithine Aminotransferase, Arseniy Butrin

Dissertations

HCC, Hepatocellular carcinoma, MBI, Mechanism-based inactivator, OAT, Ornithine Aminotransferase

The Roles Of A Probiotic Exopolysaccharide In Breast Cancer, Mai Rachel Nguyen

Dissertations

breast cancer, exopolysaccharide, probiotic

Abl2 Promotes Alcohol-Associated Liver Disease Via Ppar Gamma Regulation, Gregory Malnassy

Dissertations

Alcohol-associated liver disease (AALD) is an umbrella term for a spectrum of diseases resulting from chronic alcohol (e.g. ethanol) abuse ranging in severity from reversible conditions such as alcohol-induced steatosis to advanced and largely irreversible liver pathologies including alcoholic steatohepatitis (ASH), alcoholic hepatitis (AH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). AALD is one of the primary causes of chronic liver disease worldwide and accounts for 44% of liver disease deaths in the United States. Drinking rates, both in the United States and globally, have increased year over year for the past three decades, a trend which has resulted in significantly …

Function Of Mllt3 In Liver Metabolism, Grace Ann Hammack

Master's Theses

The chromatin regulator MLLT3 recognizes target genes through the YEATS domain that identifies post-translationally modified histones, with preference for crotonyl and acetyl marks, and recruits different multiprotein effector complexes through its C-terminal domain to target genes. To study the role of MLLT3 in gene regulation, the Zeleznik-Le and Hemenway labs developed Mllt3 whole-body knockout (Mllt3;Rosa26-CreERT2) mice. These mice have a hematopoietic stem cell phenotype and an unexpected obesity and hepatic steatosis phenotype. It was unknown whether these phenotypes were from liver intrinsic effects or influenced by other parts of the body. To study this fatty liver phenotype further, Mllt3;Alb-Cre were …

Beta-Adrenergic Receptor Mediated Transcriptional Dysregulation In Hematopoeitc Stem And Progenitor Cells Leads To Bone Marrow Erythroid Suppresion In Multiple Myeloma Patients - Ex Vivo Investigations, Vimal Ravi Subramaniam

Master's Theses

The sympathetic nervous system (SNS) regulates our body’s involuntary response to stress or harm. Once activated, it releases hormones and neurotransmitters known as catecholamines throughout the body to mediate its recovery. The catecholamines bind to adrenergic receptors located on various cells in a process designated as adrenergic signaling to elicit this response. This process is not always to our benefit. Adverse symptoms such as anemia can result due to adrenergic signaling. Our laboratory previously showed that burn injury triggers adrenergic receptor stimulation resulting in anemia induced by diminished erythropoiesis. The development of anemia in these studies was found to be …

Selectivity For Allosteric Effectors Of Bacterial Adp-Glucose Pyrophosphorylase: Structural And Functional Studies, Mashael Alghamdi

Dissertations

The biosynthesis of the intracellular polysaccharide in bacteria and plants, glycogen and starch, respectively, controlled by the key regulatory step that catalyzed by ADP-Glucose Pyrophosphorylase (ADP-Glc PPase). ADP-Glc PPase is an allosteric enzyme regulated by metabolites produced of the principle carbon assimilation pathway in each organism. Agrobacterium tumefaciens enzyme activated by fructose 6-phosphate (Fru6P) and pyruvate, whereas Escherichia coli enzyme activated by fructose 1,6-bisphosphate (FBP), and both enzymes inhibited by AMP. Here, we targeted the allosteric regulation of the A. tumefaciens enzyme, examined some residues that may impact the regulation mechanism (Ser72, His71, Arg75, Ser351, Ser334, Arg368, Asn350, and Asp291), …

Human Isocitrate Dehydrogenase Enzymes, Joseph Valentein Roman

Dissertations

Mammals have three forms of isocitrate dehydrogenase (ICDH) that each catalyze the oxidative decarboxylation of isocitrate forming alpha-ketoglutarate (AKG), carbon dioxide and reduced pyridine nucleotide. The intra-mitochondrial, citric acid cycle enzyme is ICDH III that exists as a hetero tetramer and uses NAD+ as a substrate. ICDH I and ICDH II are both dimeric enzymes that reside in the cytosol and use NADP+. While, the precise function of the cytosolic ICDHs is not known, active site variants of both ICDH I and II cause a variety of cancers. The R132H variant of ICDH1 is one of the most common alterations. …

Mllt3 Regulates Expression Of Genes Critical To Liver Metabolic Homeostasis, Jonathan Richard Dixit

Master's Theses

Hepatic steatosis (fatty liver) is increasingly prevalent in the US, and is characterized by triglyceride accumulation within hepatocytes. This is a direct result of an imbalance between lipid import, export, de novo lipogenesis, and beta oxidation. Hepatic steatosis can also progress to nonalcoholic steatohepatitis (NASH) which is characterized by increased hepatocyte damage, inflammation, and fibrosis. Further progression can lead to liver failure or hepatocellular carcinoma, requiring liver transplant or causing death. Our lab recently created Mllt3 conditional whole body knockout mice. These mice developed hepatic steatosis and NASH following Mllt3 deletion. MLLT3 is a master gene regulator that is part …

Yczr, A New Case Of Plp-Dependent Mocr/Gabr Type Transcription Regulator In Klebsiella Pneumonia, Yuanzhang Zheng

Dissertations

Increasing number of genes encoding PLP-dependent transcription regulators, MocR/GabR type regulators, have been identified in various bacterial genomes. However, only a handful of them, including MocR, PdxR and GabR have been studied experimentally. They control different aspects of the bacterial metabolism. Only GabR has reported crystallographic structures. MocR/GabR regulators possess a chimeric structure consisted of a WHTH DNA binding domain and an Aminotransferase-like regulation domain, which can bind PLP as an effector in transcription regulation. Such a chimeric construct presents an interesting case in molecular evolution. The regulation domains of All MocR/GabR type regulators loss their catalytic capacity during evolution …

Characterization Of The Effects Of Sex And Estrogen Receptor Signaling On Antigen-Specific T Cells For Immunotherapy, Flor Cecilia Navarro Negredo

Dissertations

Adoptive cell transfer (ACT) immunotherapy using antigen (Ag)-specific T cells is partially effective treating several malignancies but numerous challenges remain in order to improve its therapeutic potential. The roles of host factors, such as sex hormone receptor signaling, that can affect the T cell anti-tumor function remain understudied. The work performed in this dissertation characterized the role of estrogen signaling on T cell function in vitro, and during ACT immunotherapy against hepatocellular carcinoma (HCC) in vivo. Estrogen signaling through ERa enhanced the expression and secretion of Type I effector cytokines including IFNg, TNFa, and Granzyme B in male and female …

A Discrete Loop In Serca N-Domain Plays A Role In Serca Headpiece Dynamics And Function, Olga N. Raguimova

Dissertations

The sarco/endoplasmic reticulum calcium ATPase (SERCA) is the major regulator of Ca2+ levels in the cell. Deficient calcium handling in the heart has been linked to heart failure, a leading cause of death in developed countries. As of today, targeting SERCA to enhance cardiac function has not been successful due to lack of details about SERCA structural dynamics during Ca2+ transport.

In my research, I utilized MD simulations and variety of physical assays to determine the role of Nβ5-β6 loop in regulation of SERCA structural dynamics during Ca2+ transport. Previous MD simulations by our lab predicted that the Nβ5-β6 loop …

Comparison Between The Structure-Function Relationship In The Wild Type Gαi1 Protein And Its Oncogenic Mutant, Jesse Lee Goossens

Dissertations

Many signal transduction pathways are regulated by guanine nucleotide-binding (G?) proteins, which function as molecular switches fluctuating between active and inactive conformations. Proper function depends on three flexible switch regions that are involved in the relatively slow hydrolysis of GTP. Deep sequencing studies have found mutations in the GNAS and GNAI1 genes involved in tumorigenesis, among which include a mutation corresponding to a highly conserved arginine residue in the switch II region. A mutation in GNAI1 encoding an R208Q change in G?i1 has been linked to intestinal cancers. We investigated the molecular basis of oncogenesis of this mutant by studying …

Characterizing The Requirement Of The Cmi/Trr Compass-Like Complex During Drosophila Development, Timothy Nickels

Master's Theses

The MLR family of COMPASS-like complexes are histone methyltransferase complexes that are associated with the activation of gene enhancers. In D. melanogaster, Cara mitad (Cmi, also known as Lpt) and Trithorax related (Trr) are central subunits of a complex orthologous to mammalian Lysine methyltransferase 2 C and D (KMT2C and KMT2D, also known as MLL3 and MLL2/4) that catalyze H3K4 monomethylation. Previous studies have demonstrated that mutations in these genes are associated with cancer and developmental disorders, but the mechanisms by which these alterations contribute to disease states are unknown. The Cmi-containing COMPASS-like complex and orthologous vertebrate complexes have been …

Inhibition Of Mutant Egfr In Nsclc Promotes Endothelin-1-Mediated Nsclc Disease Progression, Stephen Ollosi

Master's Theses

Angiogenesis in NSCLC has been identified as important therapeutic target in combination with EGFR TKIs. However, only small incremental advancements have been made for the use of angiogenesis inhibitors in NSCLC and it remains elusive why the inhibition of VEGF-mediated neovascularization is not therapeutically efficacious. I present experimental evidence that a subpopulation of NSCLC cells with EGFR TKI-induced EMT contributes toward the attenuation of the response to EGFR TKI therapy. One of the hallmarks of cancer is heterogeneity and I have previously demonstrated that tumor heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations gives rise to divergent resistance …

Regulation Of Bacterial Glycogen Synthesis: Structure-Function Relationship Of Adp-Glucose Pyrophosphorylase, Hiral Priyank Patel

Dissertations

Starch is an important source for energy, and it has become a significant resource for bio-fuel production. ADP-glucose pyrophosphorylase is the enzyme that controls the synthesis of starch in plants, and glycogen in bacteria. This regulation is mainly driven by allosteric activators (Fru6P, FBP, and Pyruvate) in bacteria. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. Here we show that one specific subunit interface and the interaction between amino acids Arg11 and Asp141 are critical for the regulatory signal in the enzyme from Agrobacterium tumefaciens. Manipulation of this regulatory signal is critical …

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 …

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 …

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 …

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 …

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 …

Role Of Pkc Delta In Uv Radiation Dna Damage Repair, Gargi Patil

Master's Theses

DNA damage caused by ultraviolet radiation (UV), such as cyclobutane pyrimidine dimers (CPD), is repaired by the nucleotide excision repair (NER) pathway. When NER is defective, DNA damage is not repaired, leading to mutations and skin cancer. After DNA damage, the cell cycle is halted at various checkpoints to allow time for repair of the damage and maintain genomic integrity, however little is known about the coordination between NER DNA damage repair and cell cycle halting at checkpoints after DNA damage. Protein kinase C δ (PKCδ) plays major role in apoptosis and maintains the G2/M checkpoint in response to UV …

A Bioluminescence Sensor Of Nlrp3 Inflammasome Activation, Michael Alexander Winek

Master's Theses

The innate immune system is many organisms first line of defense against pathogenic insult or tissue damage. This defense strategy is intent on restoring homeostasis upon perturbation. Upon activation of the innate immune system in humans, an oligomeric protein complex termed the “Inflammasome” forms in myeloid cells. The canonical output of activation of any subset of inflammasome is Caspase-1-mediated secretion of pro-inflammatory cytokines IL1β and IL18. Chronic or uncontrolled inflammasome activation is at the core of myriad economically burdening diseases. In many of these diseases, endogenous factors chronically engage the innate immune system. To study these diseases in in vivo, …

The Role Of C-Abl Kinase In Hcc Development, Lennox Chitsike

Master's Theses

Hepatocellular Carcinoma is the second most lethal cancer after pancreatic cancer. Unresectable HCC tumors carry a poor prognosis and few treatment options are available. The dismal prognosis is mainly due to limited therapy options and molecularly targeted therapy is deemed as solution. Here, we report a novel role of c-Abl in HCC development. We provide evidence of c-Abl activation in human HCC samples compared to normal liver. Using genetic and pharmacological tools, we show that c-Abl plays a vital role in HCC progression in vitro and in vivo. We have identified Axl as an effector in processes mediated by c-Abl. …

Regulation Of The Subunits Of Hypoxia Inducible Factors By Sprouty2 And Its Impact On Different Biological Processes, Kristin Caroline Hicks

Dissertations

The hypoxia inducible factors (HIF1α, HIF2α, HIF1β) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation via different mechanisms. Because of the seemingly opposite actions of the HIF and Spry2 on cellular processes, we hypothesized that Spry2 decreases the protein levels of HIF1α, HIF2α and HIF1β by enhancing the proximity of the HIF subunit to an ubiquitin ligase capable of degrading the subunit. Focusing on HIF1α as a prototypical alpha subunit, in a variety of tumor derived cell lines, Spry2 decreases the protein …

Studies Into The Allosteric Regulation Of Adp-Glucose Pyrophosphorylases, Benjamin Luke Hill

Dissertations

The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site directed mutagenesis, and kinetic characterization, and protein crystallography we found a critical region for transmitting the allosteric signal in the Escherichia coli and A. tumefaciens ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize communication pathways that link allosteric and active sites, and this was tested by site-directed mutagenesis and kinetic characterization of the mutant enzymes. In addition, the application of x-ray crystallography enabled the pinpointing of the …