Biochemistry, Biophysics, and Structural Biology Commons^™

Understanding The Molecular And Cellular Functions Of Odd-Skipped Related 1 In Outflow Tract Development, Menglan Xiang

Theses and Dissertations

The cardiac outflow tract (OFT) is a transient conduit that connects the embryonic heart chambers to the vascular network. Transcription factor Osr1 promotes the proliferation and cell cycle progression of second heart field (SHF), an essential cell population that contribute to the developing OFT. In this study, we investigated the role of Osr1 in OFT development on cellular and molecular levels using a systems biology approach. We observed OFT rotation and elongation defects, as well as double-outlet right ventricle and overriding aorta as a result of SHF-specific deletion of Osr1. Using genetic inducible fate mapping, we showed that Osr1-expressing SHF …

Does Thermotolerance In Daphnia Depend On The Mitochondrial Function?, Rajib Hasan

Electronic Theses and Dissertations

Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18^ᵒC and 25^ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP …

The Gsk-3Β-Fbxl21 Axis Regulates Tcap Via Ubiquitin-Mediated Proteasomal Pathway In The Cytoplasm, Jiah Yang

Dissertations and Theses (Open Access)

Protein turnover is one of the most essential mechanisms controlling circadian rhythms. F-Box and Leucine Rich Repeat Protein21 (FBXL21) is a circadian E3 ligase which shows oscillatory mRNA transcripts and protein levels. It was previously found to perform subcellular compartment-specific E3 ligase activities targeting the core clock proteins CRYPTOCHROME(CRY)1/2. Here we identified a new sarcomeric target substrate, Telethonin(TCAP), which also shows circadian oscillation in its mRNA transcript and protein expression and, importantly, interaction with FBXL21 in an anti-phasic manner. Via computational and pharmacological tests, we identified Glycogen Synthase Kinase-3β(GSK-3β) as a regulator of FBXL21. Biochemical and molecular characterizations demonstrated that …

Antibiotic Drug Discovery Targeting Bacterial Metabolism, Miranda J. Wallace

Theses and Dissertations (ETD)

Over the last century, the use of antibiotics has enabled many advances in modern medicine, making life as we know it possible. In recent years, however, emerging bacterial resistance to virtually all major antibiotic classes has resulted in a worldwide increase in morbidity, mortality, and financial burden associated with drug resistant infections. The antimicrobial resistance crisis presents an urgent need for new antimicrobials with distinct mechanisms of action from existing drugs. The current pharmaceutical pipeline of new antibiotics is limited due to three obstacles: a lack of understanding of resistance mechanisms, a dearth of novel mechanisms of action among new …

The Distinct Expressions Of Integrins Αdβ2 And Αmβ2 Differently Regulate Macrophage Migration In 3d Matrix In Vitro And In Tissue During Inflammation, Kui Cui

Electronic Theses and Dissertations

Chronic inflammation is an essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration and accumulation of macrophages in damaged tissues. Macrophage motility is highly regulated by adhesive receptors, integrins. Namely, intermediate expression of integrin supports macrophage migration, while a high integrin density inhibits it. Our studies are focused on evaluation of the contribution of related integrins α_Dβ₂ and α_Mβ₂ to macrophage migration and development of chronic inflammation.

We found that integrin α_Dβ₂ is upregulated on M1-macrophages in vitro and …

Obscurin Mediates Ankyrin Complex Formation In The Heart, Janani Subramaniam

Dissertations and Theses (Open Access)

Distinctly organized domains of receptors, ion channels, transporters, signaling molecules, cell adhesion molecules, and contractile proteins are crucial to cardiac function. Interactions between adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play a pivotal role in organizing these functional domains in cardiomyocytes. Therefore, dysfunction of both ankyrin as well as obscurin lead to a host of cardiovascular diseases such as arrhythmias and cardiomyopathies. Alternative splicing of ankyrin yields numerous isoforms that interact with obscurin at various sub-cellular domains. And while some of these obscurin-ankyrin complexes have been studied, many others have not been characterized. Further, previous studies …

Investigations Of The Structure-Function Relationship In Kainate Receptors Using FöRster Resonance Energy Transfer, Douglas Litwin

Dissertations and Theses (Open Access)

Kainate receptors belong to the family of ion channels known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission, modulate the release of presynaptic glutamate, and facilitate dendrite formation. Kainate receptors are unique among the ionotropic glutamate receptors in being modulated by sodium ions. They have also been implicated in the development of higher learning and epilepsy. In recent years a wealth of structural data has become available for the AMPA and NMDA classes; however, the structural characterization of kainate receptors has been limited. The work in this dissertation utilizes luminescence resonance energy transfer …

Deubiquitinating Enzymes Promote Cancer Progression And Metastasis Via Regulating Protein Stability, Zhenna Xiao

Dissertations and Theses (Open Access)

Deubiquitinating enzymes (DUBs, also called deubiquitinases) are enzymes that remove monoubiquitin or polyubiquitin chains from target proteins. DUBs have critical roles in cell homeostasis and signal transduction, as they regulate protein degradation, subcellular localization, and protein-protein interaction. Deregulation of DUBs contributes substantially to tumor formation and progression, and therefore targeting DUBs may be a promising cancer therapy strategy. My dissertation focuses on identifying the DUBs of EZH2 and SNAI1, two proteins critical for cancer progression and metastasis, and establishing these DUBs as promising anti-cancer targets.

EZH2, the catalytic component of the PRC2 complex, silences gene transcription by histone methylation. High …

Dissecting The Roles Of Human Smc Complexes In Transcription Regulation And Chromatin Organization, Ruoyu Wang

Dissertations and Theses (Open Access)

Metazoans utilize a constellation of distal regulatory elements to control gene transcription, and therefore they have to forge highly complex chromatin loops to spatially bridge these regulatory elements and genes in the three-dimensional (3D) genome. However, the hierarchy of chromatin contacts and their underlying mechanisms are not well-understood. SMC complexes including Cohesin complex and Condensin complex has been widely proposed to organize 3D genome structure, and further regulate metazoans’ gene transcription. Here, we aim to dissect the direct functions of SMC complexes (both Cohesin and Condensin) in transcriptional regulation and 3D genome organization, by utilizing an inducible protein degradation system. …

Investigation Of Microbiota In Health And Disease Of Poultry, Bishnu Adhikari

Graduate Theses and Dissertations

The microbiotas play vital roles in health and diseases of both humans and animals. 16S rRNA genes sequence analysis is one of the most popular and commonly used methods in the analysis of microbiotas associated with hosts. In this dissertation, the microbiotas of chickens (broilers, breeders, and layers) and turkeys were evaluated by 16S rRNA gene sequencing. Characterization of the culturable subpopulations of Lactobacillus in the chicken gut can serve as a valuable resource for probiotic development. In Chapter 2, Lactobacillus subpopulations recovered on MRS from chicken gut were defined comprehensively for the first time using 16S rRNA gene profiling, …

Translational Control Of Antibiotic Resistance, Anne Witzky, Rodney Tollerson Ii, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Many antibiotics available in the clinic today directly inhibit bacterial translation. Despite the past success of such drugs, their efficacy is diminishing with the spread of antibiotic resistance. Through the use of ribosomal modifications, ribosomal protection proteins, translation elongation factors and mistranslation, many pathogens are able to establish resistance to common therapeutics. However, current efforts in drug discovery are focused on overcoming these obstacles through the modification or discovery of new treatment options. Here, we provide an overview for common mechanisms of resistance to translation-targeting drugs and summarize several important breakthroughs in recent drug development.

Intrinsically Disorder Proteins And Liquid-Liquid Phase Transitions In Neurodegenerative Diseases, April Lynn Darling

USF Tampa Graduate Theses and Dissertations

Neurodegenerative diseases have a negative impact on health and economics, effecting more than 20 million people in the United States and costing around $800 billion in 2018. Additionally, most neurodegenerative diseases such as Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), and several others are fatal and have no cure or effective treatment. Many of these diseases have a common theme of protein aggregates being present in disease tissue. These aggregates are thought to be toxic to the cell and prevention of pathological aggregation and elimination of these cellular aggregates may serve as a potential therapeutic approach to …

Novel Strategies On Characterizing Biologically Specific Protein-Protein Interaction Networks, Bi Zhao

USF Tampa Graduate Theses and Dissertations

The function, behavior, and environmental response of biological systems are essentially determined by the complex interaction and regulation of biomolecules inside the systems. Therefore, it is critical to characterize the inter-molecular interaction and regulation of biomolecules inside these systems. In this direction, many experimental techniques have been developed and these techniques have been used in many different model systems under various conditions. Consequently, a massive amount of data has been generated. These data cover multiple aspects of molecular interaction and regulation, such as protein-protein interaction, microRNA-RNA interaction, gene expression profiles, etc. While carrying rich information, these data may also contain …

Targeting Pten For Therapy In Cancer And Ptenopathies, Emily Palumbo

USF Tampa Graduate Theses and Dissertations

PTEN, a dual protein and lipid phosphatase, regulates a myriad of cellular functions including PI3K pathway signaling, cell migration, proliferation, invasion and apoptosis. PTEN mutations often lead to multiple malignancies, including prostate, breast, endometrial, skin and brain cancers, associated with hyperactive PI3K signaling. PTEN mutations have also been associated with a variety of other diseases, classified as PTEN Hamartoma Tumor Syndromes (PHTS). In addition, compromised function or reduced expression of PTEN due to non-genomic mechanisms are associated with many types of hyperproliferative diseases, such as restenosis and neoplastic diseases, including melanoma, lung, breast, prostate and colon cancers. Although PI3K pathway …

Roux-En-Y Gastric Bypass Attenuates Obesity-Related Liver Injury, Drew Alan Rideout

USF Tampa Graduate Theses and Dissertations

Obesity is a growing epidemic in the United States with significant co-morbidities. Non-Alcoholic Fatty Liver Disease (NAFLD) is a prevalent manifestation of obesity that can lead to cirrhosis. Roux-en-Y gastric bypass (RYGB) results in substantial long-term weight loss and resolution of obesity-related metabolic diseases. There appears to be a weight-independent molecular mechanism for the improvement of diabetes mellitus and NAFLD after RYGB, which is poorly understood. Obesity is associated with chronic inflammation that accompanies the hepatic steatosis. Through unknown mechanisms, RYGB in humans increases serum levels of the fat-derived adipocytokine, adiponectin. Adiponectin (an anti-inflammatory cytokine) is known to have a …

A High Throughput Assay For The Detection Of Stimulator Of Interferon Genes (Sting) Agonists, Michael J. Ingling

Graduate School of Biomedical Sciences Theses and Dissertations

The innate immune system includes a menagerie of different cell types, each with a different role in the process of monitoring the body for invaders and presenting gathered debris (antigen) to the adaptive immune system. Somatic cells have intracellular receptors for the same purpose. Cancer cells, however, have avoided these methods of detection despite, in many cases, the tumor’s immunogenic traits. Immuno-oncology is a field dedicated to the immunological traits of tumors, more recently finding ways of instigating an immune response against tumors. In this regard, STING, a receptor of cyclic dinucleotides (CDN), has come to the forefront of immuno-oncology. …

Characterization Of Human Dutpase, Shawna Marie Rotoli

Graduate School of Biomedical Sciences Theses and Dissertations

Deoxyuridine nucleotidyl transferase (dUTPase) is an enzyme found in all organisms that have thymine as a component of DNA. It catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate thus precluding the buildup of dUTP pools as well as providing the substrate, dUMP, for the de novo synthesis of thymidylate. In Homo sapiens, there are four isoforms: mitochondrial (mDut), nuclear (nDut), variant 3 and variant 4. This work is largely focused on nDut. Using structural and MS analyses of recombinant dUTPase constructs, an intermolecular disulfide bridge between cysteine-3 of each nDut monomer was discovered. It was found that these two …

The Expression Of Connexin-43 By Cd11c+ Dendritic Cells Is Required To Maintain Cd4+ Foxp3+ Regulatory T Cell Population In Peripheral Lymphoid Organs, Caroline Titus Miller

Biological Sciences Theses & Dissertations

Foxp3⁺ regulatory T cells (T_R) are an immunosuppressive subset of CD4⁺ T cells that maintain homeostasis of the immune system. They are sustained by the interaction between the Major Histocompatibility Complex (MHC) molecules present on antigen presenting dendritic cells and the T Cell Receptor (TCR) expressed on T_R cells that is specific for the MHC loaded with an antigenic peptide. Here, we show that in addition to MHC/TCR interaction, Connexin-43 (Cx43) expression by dendritic cells is required to maintain the TR cell population. CD11c⁺ dendritic cells represent a major subset of antigen presenting cells. …

Endogenous Force Transmission Between Epithelial Cells And A Role For Α-Catenin, Sandeep Dumbali

Mechanical & Aerospace Engineering Theses & Dissertations

In epithelial tissues, epithelial cells adhere to each other as well as to the underlying extra-cellular matrix (ECM). E-cadherin-based intercellular junctions play an important role in tissue integrity. These junctions experience cell-generated mechanical forces via apparent adaptor proteins such as beta (β) catenin, alpha (α) catenin and vinculin. Abnormalities in these junctions may result in skin related diseases and cancers. Here, I devised methods to determine the endogenous intercellular force within cell pairs as well as in large epithelial islands. I further ascertained the factors that affect the level of inter-cellular tension.

Experiments with pairs of epithelial cells exogenously expressing …

Characterizing Novel Pathways For Regulation And Function Of Ataxin-2, Elise Spencer Melhado

Theses and Dissertations

Ataxin-2 is an RNA-binding protein that is involved in many crucial cellular processes such as R-loop regulation, mRNA stability, TOR signaling regulation, and stress granule formation. Ataxin-2 is highly conserved, found in organisms ranging from Saccharomyces cerevisiae to Caenorhabditis elegans and Homo sapiens. Recently, ataxin-2 has been linked to the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal disease that causes loss of motor neurons. In addition to ataxin-2 interacting with known ALS risk factor proteins, research into the relationship between ataxin-2 and ALS shows that polyglutamine expansions in ataxin-2 are gain-of-function mutations that lead to overactivity of …

Snfing Glucose To Pass Mitochondrial Dysfunction: The Role Of Two Sensory Protein Kinases In Metabolic Diseases, Kai Li Ong

Theses and Dissertations

Mitochondria is no longer viewed as merely a powerhouse of the cell. It is now apparentthat mitochondria play a central role in signaling, maintaining cellular homeostasis and cell fate.Mitochondrial dysfunction has been linked to many human diseases caused by cellular metabolicderegulation, such as obesity, diabetes, neurodegenerative disease, cardiovascular disease andcancer. Eukaryotic organisms have evolved an efficient way in sensing, communicating andresponding to cellular stress and regulating mitochondrial activity correspondingly through acomplex network of intercommunicating protein kinases and their downstream effectors. Thisdissertation focuses on the interplay of two of the master metabolic regulators in the cell: AMPKand PASK, and characterization of …

Knocking Out A Negative Regulator Of Hedgehog Signaling Blocks Differentiation Of Cells Into Neurons, Danielle Margaret Spice, Gregory M. Kelly Ph.D.

Western Research Forum

Hedgehog (Hh) signaling, one of many different protein signaling pathways found in mammals, is vital in many stage of neural development. A major negative regulator of Hh signaling is a protein known as Suppressor of Fused (SUFU), which acts to sequester the full length Gli transcription factors, proteins that can turn genes on and off, in the cytoplasm or facilitates its conversion to a repressive form. The P19 embryonal carcinoma cell line is a model of hind-brain neuronal differentiation and the involvement of Hh signaling, in particular the role of SUFU in this process has yet to be explored. We …

Iron-Sulfur Cluster Biosynthesis In Methanogens, Cuiping Zhao

LSU Doctoral Dissertations

Methanogens live in a syntrophic consortium with bacteria, taking advantage of the metabolic abilities of their syntrophic partners to overcome energetic barriers and break down compounds that they cannot digest by themselves. Interspecies electron transfer, which is a major type of microbial communication in syntrophic processes, improves methanogenesis and anaerobic oxidization of methane (AOM) processes involved in syntrophic consortia. These processes have a significant impact on the global carbon cycle. Most of the essential enzymes involved in methanogenesis are iron-sulfur proteins. Iron-sulfur clusters are one of the oldest and most versatile cofactors present in all domains of life. To date, …

Functional Characterization Of The Ovarian Tumor Domain Deubiquitinating Enzyme 6b, Jasmin M. D'Andrea

USF Tampa Graduate Theses and Dissertations

The posttranslational modification ubiquitination is major regulatory mechanism used throughout cell signaling pathways such as cell cycle regulation and the DNA damage response. As such, the E3 ligases and their deubiquitinating enzyme counterparts, which conjugate and deconjugate ubiquitin to and from protein substrates respectively, must be tightly regulated to prevent aberrant cellular behaviors that could lead to diseases such as cancer.

Of the five families of deubiquitinating enzymes, the Ovarian Tumor Domain (OTU) family is fairly unique and under-studied; many of its family members hold a linkage specificity to certain ubiquitin chains and a number of them have been implicated …

Divergent Transcriptional Regulation Of Suppressors Of Cytokine Signaling Genes In Adipocytes, Paula Mota De Sa

LSU Doctoral Dissertations

The Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway transduces several signals crucial for development and homeostasis. Suppressors of cytokine signaling (SOCS) proteins control JAK-STAT signaling via a negative feedback loop. The transcription factor STAT5 is known to play a significant role in fat cell development and function, and several studies suggest that acetylation may affect STAT5 transcriptional activity. To test this hypothesis, we treated 3T3-L1 adipocytes with growth hormone (GH) to activate STAT5 in the presence or absence of histone deacetylase (HDAC) inhibitors. STAT5 acetylation levels were low in adipocytes and mostly unchanged by the …

In Vitro Evaluation Of Ovarian Cancer Tumorigenesis As A Function Of Quinone Oxidoreducatse-1 And Cell Phenotype, Milcah S. Jackson

LSU Doctoral Dissertations

In vitro multicellular spheroids are attractive model systems for assessing genetic and epigenetic changes that occur in diseased tissues. Understanding how such alterations in gene and subsequent protein expression affect disease progression and metastasis, drug resistance, and recurrence is of great interest in cancer research. In this regard, examining expression and activity of proteins, such as those with cytoprotective ability that are overexpressed in cancer cells, in addition to cell phenotype (i.e., stem-like, epithelial, mesenchymal, or mixed), are two ways to evaluate genetic and epigenetic changes. Moreover, determining the impact that cytoprotective proteins and cell phenotype have on tumor formation …

Gαi And Gɣ30a Act Downstream Of Tre1 In Drosophila Courtship, Emily Mu

McNair Research Journal SJSU

The role of genes in morphological development is well understood for a variety of model organisms, but there remains a gap in our understanding of how genetics mediate behavior. Are there master genes that regulate behavior? Answering this question will lead to a better understanding of the development and function of the central nervous system, eventually allowing us to map out the pathways that regulate specific behaviors. We are using Drosophila melanogaster as a model organism and the male courtship ritual as the behavior of interest to study the relationships between genes, neural development, and behavior. Trapped in endoderm 1 …

Insulin Signal Transduction Mediates Ethanol-Induced Feeding Dysfunction In A Fly Model Of Fetal Alcohol Spectrum Disorder, Manae Matsubara

McNair Research Journal SJSU

Fetal alcohol spectrum disorder (FASD) is the leading cause of congenital intellectual disabilities in the Western World, with a worldwide prevalence of 2-11% of all births. FASD is preventable but recent epidemiological studies suggest that public awareness campaigns have reached the limits of their effectiveness. Therefore, research is shifting from prevention to treatment and mitigation of symptoms. No biological treatments for FASD exist, due in part to the fact the cellular mechanisms of alcohol toxicity are not well-understood. Developmental alcohol exposure (DAE) causes a variety of deleterious effects in both vertebrates and invertebrates, including increased mortality, slow growth, learning and …

Application Of Single Particle Electron Microscopy To Native Lens Gap Junctions And Intrinsically Disordered Signaling Complexes, Janette Bernadette Myers

Dissertations and Theses

Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part …

The Effect Of Intracellular Zinc Levels In Escherichia Coli On Metallo-Β-Lactamase Activity, Jill Thompson

Graduate School of Biomedical Sciences Theses and Dissertations

Antibiotic resistance is a consequence of evolution via natural selection and poses a serious threat to the world's population. Escherichia coli, and other bacterial species, have acquired genes encoding Metallo-β-Lactamases (MBLs). MBLs are zinc-dependent enzymes that degrade most β-lactam antibiotics, and have emerged as important antibacterial resistance determinants in multidrug resistant pathogens. There are several different families of MBLs, and the major clinically relevant ones are Verona Integrated-encoded Metallo-β-Lactamases (VIMs), Imipenemases (IMPs), and New Delhi Metallo-β-Lactamases (NDMs). These enzymes confer high level resistance to penicillins, cephalosporins, and carbapenems, and are not inhibited by FDA-approved β-lactamase inhibitors. Variants of NDM have …