Life Sciences Commons^™

Purification Of Recombinant E. Coli Topoisomerase Iii For Structure-Based Drug Design Using Protein Crystallization, Miguel A. Perez Rodriguez, Yuk-Ching Tse-Dinh

FIU Undergraduate Research Journal

Type IA Topoisomerases are ubiquitous enzymes found throughout all life forms and species. These topoisomerases relieve the topographical constrains formed by DNA during processes like replication and transcription via a cleavage-religation mechanism performed through a catalytically active tyrosine residue in the primary structure of the enzyme. E. coli Topoisomerase III (EtopIII) is a type of Type IA topoisomerase, and its main function in the cell is as a decatenase, which means that it unlinks circular or intertwined pieces of genetic material and creates two unlinked segments of DNA from a singular linked chain. Structure-based determination of the enzyme’s three-dimensional structure …

Assay Replicability In Β-Glucosidase Enzyme Kinetics Across Laboratories, Nicole-Tia Najam, Jaime Mayoral

FIU Undergraduate Research Journal

Replicability is the foundation of research in any scientific discipline. Despite this fact, few studies address experimental variability within and across multiple institutions that operate under the same protocol. While consistency is usually well documented within the same lab, multi-institutional experiments may introduce new variables and, therefore, variability that may lead to inconsistent results. This study seeks to explore intra- and interinstitutional variability among enzyme catalytic efficiency values (K_M and K_cat/K_M) for the wild type of β-Glucosidase derived from Paenibacillus polymyxa. A standardized protocol for the assay was provided to all institutions that participated in …

Body Fluid Identification Using Dna Extraction Waste Product, Rachel M. Tolces

FIU Electronic Theses and Dissertations

In many situations, the amount of DNA evidence recovered at a crime scene is at trace levels, limiting the amount of testing that can be done on the evidence. Because genetic profiling is generally considered the most imperative assay to be completed on a DNA sample, in situations where yield is low, DNA extracts may not be utilized for any other purpose. Fortunately, an alternate source of DNA may exist by utilizing the waste products resulting from DNA extraction.

The goal of this project was to a protocol for recovery of DNA from robotic extraction waste and utilizing this DNA …

Serpin-Derived Novel Peptide For The Treatment Against Hiv-Induced Inflammation In The Central Nervous System, Yemmy Soler

FIU Electronic Theses and Dissertations

In the brain, HIV predominantly infects microglia/macrophages and astrocytes to a lesser extent. These cells form virus reservoirs with low levels of infection that are very hard to eradicate. Even though the use of cART increases survival rate in HIV patients, the virus persists as a chronic condition. cART is not able to effectively cross the BBB, control HIV replication, or attenuate inflammation in brain reservoirs. Therefore, the virus still causes neuronal dysfunction, pain-related pathology, and ultimately HAND. In this study, we decided to test the hypothesis that a serpin-derived small peptide, SP16, can serve as an anti-viral, anti-inflammatory, pro-survival, …

An Investigation To Corroborate Volatile And Biological Profiles Of Human Odor For Forensic Subject Identification, Chantrell J.G. Frazier

FIU Electronic Theses and Dissertations

Hands are an integral part in transferring complex microbial communities to and from our surroundings. As a result, hands are significant in provenance investigations as specific microbiota can be deposited on everyday objects through touch interactions. Skin microbiome, including bacteria, fungi, and viruses, are unique to each person, and this ‘uniqueness’ can be exploited and applied to forensic identification. Skin microbiota and volatile organic compounds (VOCs) are closely related due to specific bacteria breaking down non-volatile organic compounds to volatile organic compounds that are characteristically present in human scent. However, analyses of microbiota from touch interactions have proven to be …

Aptamer-Based Voltammetric Biosensing For The Detection Of Codeine And Fentanyl In Sweat And Saliva, Rosa Lashantez Cromartie

FIU Electronic Theses and Dissertations

Despite the many governmental and medicinal restrictions created to combat the opioid epidemic in the United States, opioid abuse and overdose rates continue to rise. The development of an aptamer-based voltammetric sensor and biosensor is described in this dissertation. The aim was to develop a low-cost, sensitive, and specific aptamer-based sensor for on-site, label-free determination of codeine and fentanyl in biological fluids. To do this, the surfaces of screen-printed carbon electrodes (SPCE) were modified with gold nanoparticles (AuNPs), followed by the addition of single-stranded DNA aptamers. These were covalently bound to the electrode surface. Operations of the sensors were collected …

Mechanisms Of Chloroperoxidases-Catalyzed Enantioselective Transformations From Spectroscopic And X-Ray Crystallographic Studies Of Enzyme-Substrate Complexes, Xiaoqing Tang

FIU Electronic Theses and Dissertations

The chloroperoxidase secreted from Caldariomyces fumago catalyzes broad spectrum of reactions. The crystallography combined with X-ray diffraction analysis was conducted to reveal recombinant CPO expressed in a modified Aspergillus niger system. Our results indicated that despite functional similarities with wild type CPO, recombinant CPO is over glycosylated with more mannose. Besides, ten iodide ion binding sites were identified in rCPO and six of them were found to be well superimposed on previously reported structure of the wild type CPO. Therefore, recombinant CPO shares almost the same structure with wild type CPO, and the Aspergillus niger is a potential system for …

A Study Of The Mammalian High Mobility Group Protein At-Hook 2 (Hmga2) And Its Interactions With Dna, Linjia Su

FIU Electronic Theses and Dissertations

The mammalian high-mobility-group protein AT-hook 2 (HMGA2) is a small DNA-binding protein and consists of three positively charged “AT-hooks” and a negatively charged C-terminal motif. It is a multifunctional nuclear protein linked to obesity, human height, stem cell youth, human intelligence, and tumorigenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti‐obesity drugs through inhibiting its DNA‐binding activities. Here a miniaturized, automated AlphaScreen ultra‐high‐throughput screening assay is developed to identify inhibitors targeting HMGA2‐DNA interactions. After screening the LOPAC1280 library, several compounds are identified that strongly inhibit HMGA2‐DNA interactions including suramin, a negatively charged antiparasitic drug. …

Oxidative Dna Damage Modulates Genome And Epigenome Integrity Via Base Excision Repair, Pawlos S. Tsegay

FIU Electronic Theses and Dissertations

Oxidative DNA damage is one of the leading causes of genome instability, cell death, and diseases. It is repaired by DNA base excision repair (BER), during which repair and translesion DNA polymerases may incorporate damaged nucleotides and mediate RNA-guided DNA repair induced by DNA replication and gene transcription leading to the modulation of genome stability. On the other hand, oxidative DNA damage may result in cellular epigenetic responses to regulate DNA repair, altering genome stability and integrity. In this dissertation, we revealed the molecular mechanisms underlying the misincorporation of oxidized nucleotides, 5′,8-cyclo-2-cyclodeoxyadenosine (cdA) and RNA-guided base lesion repair mediated by …

High And Low Toxin Producing Strains Of Karenia Brevis Differ Significantly In The Redox Proteome, Lipid Profiles, And Xanthophyll Cycle Pigments, Ricardo Colon

FIU Electronic Theses and Dissertations

The dinoflagellate Karenia brevis, blooms annually in the Gulf of Mexico, producing a suite of neurotoxins known as the brevetoxins. The cellular toxin content of K. brevis, however, is highly variable between or even within strains. I investigated biochemical differences between high (KbHT) and low (KbLT) toxin producing cultures both derived from the Wilson strain, related to energy-dependent quenching (qE) by photosystem II, and the content of reduced thiols of the proteome. By characterizing the xanthophyll content of the two strains I was able to determine that KbLT performs qE inconsistently. To investigate the …

Type I Topoisomerases As Potential Targets For Therapeutics, Ahmed Seddek

FIU Electronic Theses and Dissertations

DNA topoisomerases are universal enzymes that control the topological features of DNA in all forms of life. This study aims to find potential inhibitors of some of the DNA topoisomerases in bacteria and humans that can be developed into potential therapeutics.

The first aim of this study is to find potential inhibitors of bacterial topoisomerase I that can be developed into antibiotics. There is an urgent need to develop novel antibiotics to overcome the world-wide health crisis of antimicrobial resistance. Virtual screening and biochemical assays were combined to screen thousands of compounds for potential inhibitors of bacterial topoisomerase I. NSC76027 …

Identification Of Human Topoisomerase I Poison And Catalytic Inhibitors, Christian Madeira

FIU Electronic Theses and Dissertations

Inhibition of human topoisomerase I have been shown to reduce excessive transcription of PAMP-induced genes based on prior studies, which offers a solution to offset complications of sepsis such as tissue damage and organ failure. The enzyme resolves the topological constraints on DNA that encodes these genes. The aim of this study is to identify human topoisomerase I (HTop1) inhibitors that can a.) prevent the relaxation of negative supercoiled plasmid DNA by HTop1 and b.) reduce HTop1 binding to DNA and formation of covalent cleavage complex (HTop1cc) utilizing a novel yeast screening system. Top hits from in-silico screening conducted by …

Bacterial Mechanisms Of Toxicity And Resistance To Organoarsenicals, Luis D. Garbinski

FIU Electronic Theses and Dissertations

Arsenic is a toxic element prevalent in the environment since the origin of life on Earth. Bacteria evolved in an arsenic-rich environment, where they developed ways to both overcome arsenic toxicity and harness it to compete with other organisms. These mechanisms include chemical modifications (e.g. oxidation, methylation), degradation, and efflux. The goal of this dissertation is to better characterize these mechanisms, illuminating the arsenic biogeocycle and allowing us to harness organoarsenical toxicity for novel antibiotics. A goal of my research was to elucidate the antibiotic properties of MAs(III), which is synthesized by bacteria to thrive over other bacteria, by identifying …

Development Of A Dna Methylation Multiplex Assay For Body Fluid Identification And Age Determination, Quentin Gauthier

FIU Electronic Theses and Dissertations

For forensic laboratories, the determination of body fluid origin of samples collected at a crime scene are typically presumptive and often destructive. However, given that in certain cases the presence of DNA is not in dispute and rather where the DNA came from is of primary concern, new methodologies are needed. Epigenetic modifications, such as DNA methylation, affect gene expression in every cell of every mammal. These DNA methylation patterns typically are observed as the addition of a methyl group on the 5’ carbon of a cytosine followed by guanine (CpG). Methylation patterns have been observed to change in response …

Structure Function Relationship In Hexacoordinate Heme Proteins: Mechanism Of Globin X Interactions With Exogenous Ligands And Ligand Accessiblity In Cytoglobin And Neuroglobin, Ruipeng Lei

FIU Electronic Theses and Dissertations

Cytoglobin (Cygb), neuroglobin (Ngb), and globin X (GbX) belongs to recently discovered members of the vertebrate globin family, they carry a heme prosthetic group that can reversibly bind exogenous ligands such as CO, NO and O₂. Although the physiological functions of Cygb, Ngb and GbX are still under debate, several possible physiological functions for these proteins were proposed. Cytoglobin was reported to participate in lipid-based signaling and to stabilize the tumor suppressor p53 upon DNA damage, which imply its anti-cancer role. Neuroglobin was shown to interact with α-subunit of the heterotrimeric G protein as well as cytochrome c …

Dna Base Excision Repair Modulates Dna Repeat Instability And Non-B Form Dna Structures, Eduardo E. Laverde

FIU Electronic Theses and Dissertations

The human genome is constantly attacked by endogenous and exogenous sources of DNA damage that generates DNA base lesions and strand breaks leading to genome instability, cell death, and diseases. To combat these adverse effects, cells have evolved a robust DNA repair mechanism called “the DNA base excision repair (BER),” which efficiently removes DNA lesions maintaining genome stability. However, its underlying molecular mechanisms remain to be elucidated. In my dissertation research, I explored the molecular mechanism by which BER modulates trinucleotide repeats (TNR) by processing non-B form structures such as hairpins and R-loops through the coordination among BER enzymes and …

Dual Role Of Rin1 In Cancer Cell Behavior: Is Cortactin A New Rin1-Interacting Partner?, Wei Zhang

FIU Electronic Theses and Dissertations

Growth factors play an essential role in abnormalities in both intracellular trafficking and signal transduction pathways responsible in normal and cancer cells. Growth factors, such as epidermal growth factor, represent a main course on the activation of mitogenic signal that contribute to the mitogen activated protein kinase (MAPK) pathway affecting cell proliferation, which is driven by Ras GTPases. However, it also induces a profound morphological change by reorganization actin and other cytoskeleton proteins, which are driven by other GTPases (i.e., Rho and Rac). Ras interference 1 (Rin1) is a key cytosolic protein, that regulates both membrane trafficking and signaling pathways …

The Role Of Inositol Polyphosphate-4-Phosphatase Type Ii B (Inpp4b) In Obese Models And Endocrine Cancers, Manqi Zhang

FIU Electronic Theses and Dissertations

INPP4B is a dual-specificity phosphatase and a tumor suppressor in prostate and breast cancers. Progression of the prostate and breast cancers depends on the androgen receptor (AR) or estrogen receptor alpha (ERα) signaling, respectively. In this work we demonstrated that INPP4B reprograms ERα transcriptional activity in breast cancer. INPP4B maintains expression and protein levels of progesterone receptor (PR), an ERα direct target gene required for mammary gland development. Consistently we demonstrated that Inpp4b knockout severely impairs lateral branching in the mammary gland of maturing virgin females. In advanced prostate cancer, activation and transcriptional reprogramming of AR frequently coincides with the …

Functionalized Aptamers For Detection Of Small-Molecule Targets, Haixiang Yu

FIU Electronic Theses and Dissertations

Aptamers have recently gained considerable attention for small-molecule detection in diverse applications such as drug identification, medical diagnostics, and environmental monitoring. However, the performance of aptamer-based sensors has been greatly limited by the low target affinity and responsiveness of small-molecule binding aptamers. This dissertation describes several novel aptamer engineering and isolation strategies to remedy this problem. Specifically, we first develop a generally applicable strategy to engineer split aptamers containing two binding domains termed cooperative-binding split aptamers (CBSAs). CBSAs exhibit higher target binding affinity and are far more responsive in terms of target-induced split aptamer assembly compared to single-domain parent split …

Topoisomerase And Tyrosyl-Dna-Phosphodiesterase Ratio As An Indicator For The Response Of Glioblastoma Cancer To Topoisomerase Targeting Anticancer Drugs, Wenjie Wang

FIU Electronic Theses and Dissertations

Glioblastoma (GBM) patients have an estimated survival of ~15 months, with the standard of care (surgery, radiation, and chemotherapy) that has only modestly enhanced patient survival. Identifying biomarkers representing vulnerabilities in GBM biology may allow for the selection of effective and safe chemotherapy options. Irinotecan (IRT), a genotoxic compound currently in clinical trials for GBM, targets topoisomerase I (TOP1) by forming an irreversible ternary DNA-TOP1 cleavage complex (TOP1cc) and leads to apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that rescues TOP1cc and reduces the effectiveness of IRT. In the current study, we evaluate the value of the …

Quantifying Anthropogenic Indicators And Changes In Dissolved Organic Matter In Coastal Urban Aquatic Ecosystems Exposed To High Tidal Flooding, Gonzalo E. Eyzaguirre

Department of Biological Sciences

Sea-level rise is causing an increase in tidal flooding in coastal urban areas. Extreme high tides, also known as king tides, are peak tide moments in which tidal amplitude is increased and shallow groundwater flows from the underlying water table are introduced. During tidal flooding in urban areas, accumulated anthropogenic indicators of different water sources are released from groundwater to surface waters, but how these tidal events affect the contributions of different water sources to urban flood waters is uncertain. We quantified tracers of anthropogenic origin including fluoride, fecal coliform bacteria, as well as dissolved organic carbon (DOC) concentrations and …

Superoxide Dismutase C Modulates Macropinocytosis And Phagocytosis In Dictyostelium Discoideum, Cong Gu

FIU Electronic Theses and Dissertations

Macropinocytosis and phagocytosis, two actin-dependent and clathrin independent events of endocytosis, enable the cells such as macrophages and neutrophils to either internalize pathogens and initiates the human innate immune response or serve as a direct entry route for productive infection of pathogen. Dictyostelium discoideum, soil-living amoeba, a unicellular eukaryote that could professionally internalize fluid phase or particles several folds more than that of macrophages and neutrophils. Additionally, multiple key signaling pathways are conserved between Dictyostelium and mammalian cells, including pathways affecting small GTPases Ras and Rac and their downstream effectors, and F-Actin remodeling. All these traits makes Dictyostelium an …

Aptamer-Based Assay For Detection Of Ochratoxin A, Amanda Nicole Bartley

FIU Electronic Theses and Dissertations

Ochratoxin A (OTA) is a potent mycotoxin found in a wide range of agricultural products that has been linked to mitochondrial damage and renal disease. The standard methods for OTA analysis currently rely on the use of high-performance liquid chromatography (HPLC) coupled to fluorescence detection or mass spectrometry. Toward a highthroughput analysis of OTA, a single-stranded DNA aptamer, modified with a fluorophore, coupled to a complementary sequence, modified with a FRET-based quencher that dissociates in the presence of the target toxin, is proposed. In order to integrate “target trapping,” aptamer immobilization methods were explored to mediate interference issues. Assays were …

Investigating The Role Of The Proximal Cysteine Hydrogen Bonding Network And Distal Pocket In Chloroperoxidase, Elwood Kwong Lam

FIU Electronic Theses and Dissertations

Chloroperoxidase (CPO) is one of the most versatile heme enzyme isolated from the marine fungus, Caldariomyces fumago. Functionally, CPO can catalyze four types of reactions: peroxidation (peroxidase-like), dismutation (catalase-like), halogenation (halogenase-like), and peroxygenation (P450-like). Structurally, CPO has a distal and proximal pockets that can be best described as a hybrid of classical peroxidase and P450s. As a heme-thiolate protein, CPO contains the conserved proximal Pro28-Cys29-Pro30 stretch found in other members of the family. However, the structural and functional roles of these proline residues remain poorly understood. Site-directed mutagenesis was undertaken to generates three CPO mutants, P28A-, P30A-, P28A/P30A-CPO. The replacement …

Mycobacterium Tuberculosis Inhibitors: Action And Resistance, Pamela K. Garcia-Moreno

FIU Electronic Theses and Dissertations

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, has been a global health problem for years. The emergence of drug resistance in this organism generates the necessity of exploring novel targets and developing new drugs. Topoisomerases are enzymes found in all kingdoms of life responsible for overcoming the topological barriers encountered during essential cellular processes. The genomes of mycobacteria encode only one type IA topoisomerase (MtopI), which has been validated as a novel TB drug target. The goal of this study is to obtain new information on the mechanism and resistance of endogenous and synthetic inhibitors of MtopI.

Rv1495 is …

Modulating Adipogenesis: Key Role Of Ras-Related Protein Rab5 And Its Effectors, Yongjun Huang

FIU Electronic Theses and Dissertations

The formation of adipocytes is a complicated process in which insulin and IGF-1 signaling pathways and numerous transcription factors control the conversion of precursor cells to mature fat cells. The Rab5 protein acts as a rate-limiting protein during receptor-mediated endocytosis by switching between a GDP-bound inactive form and a GTP-bound active form. The inactivation and activation of Rab5 are regulated by several Rab5 GTPase activating proteins (GAPs) and Rab5 guanine nucleotide exchange factors (GEFs), respectively.

This dissertation demonstrated that the activity of the small GTPase Rab5 and its regulators are essential for the differentiation of 3T3-L1 pre-adipocytes. Specifically, it showed …

Impact Of San-Mediated Signaling On Glioblastoma And Neuroblastoma Metabolism, Monica Rodriguez Silva

FIU Electronic Theses and Dissertations

Glioblastoma (GBM) is the most common and aggressive type of brain cancer, with an average life expectancy of 15 months. The standard of care for GBM, surgery accompanied by radiation and chemotherapy (temozolomide-TMZ), has not changed in over 10 years illustrating the need for new and efficacious treatments. Therefore, it is imperative to improve our knowledge of GBM physiology to understand the mechanisms driving recurrence and chemoresistance so that more effective therapeutic options can be developed. Mitochondria-cell communication is key to monitor and maintain both mitochondrial and cellular health, and signaling events on the outer mitochondrial membrane (OMM) have emerged …

Structure And Mechanism Of Mycobacterial Topoisomerase I, Nan Cao

FIU Electronic Theses and Dissertations

The enzyme DNA topoisomerase I is an essential enzyme that plays an important role in eukaryotic and prokaryotic cellular processes such as DNA replication, transcription, recombination and repair. Mycobacterium tuberculosistopoisomerase I (MtTOP1) is a validated drug target for antituberculosis treatment. Mycobacterial topoisomerase I regulates the topological constraints in chromosomes and helps in maintaining the growth of mycobacteria. The N- terminal domain (NTD) of mycobacterial topoisomerase I contains conserved catalytic domains that along with the active site Tyrosine are involved in cleaving and rejoining a single strand of DNA. Magnesium is required in DNA cleavage activity of type IA topoisomerases. …

Trinucleotide Repeat Instability Modulated By Dna Repair Enzymes And Cofactors, Yaou Ren

FIU Electronic Theses and Dissertations

Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find …

The Cyclodextrin-Perfluorinated Surfactant Host-Guest Complex: Fundamental Studies For Potential Environmental Remediation And Therapeutic Applications, Mary J. Errico

FIU Electronic Theses and Dissertations

Perfluoroalkyl substances (PFASs) are contaminants of emerging concern, and have been detected in drinking water, wildlife, humans, and the environment. Cyclodextrins (CDs), cyclic sugars composed of glucose monomers, are proposed as a potential remediation strategy. CDs can form host-guest complexes with hydrophobic molecules; this complexation could be capitalized on for PFAS removal and sequestration. These dissertation projects aim to study the fundamental host-guest interactions between a variety of PFASs and CDs for eventual applications in environmental and biological remediation. 1D and 2D Nuclear magnetic resonance (NMR) spectroscopic methods were employed to determine the strength, dynamics, and structure of the CD:PFAS …