Molecular Biology Commons^™

The Role Of Conformational Changes In Viral And Bacterial Protein Functions, Md Lokman Hossen

FIU Electronic Theses and Dissertations

Proteins do versatile work in cells. They require a cascade of structural changes to perform different tasks like binding to the other neighboring biomolecules, transporting small chemicals, activating a chemical reaction, etc. The structural conformations of proteins can be critical in changing their working ability. In this dissertation, I investigated the role of conformational changes of viral protein, e.g., spike and envelope protein of SARS-CoV-2, and bacterial protein, e.g., multidrug transporter and toxic extrusion protein- PfMATE from Pyrococcus furiosus. Also, I performed molecular docking-based drug screening targeting the E protein to suggest a set of drugs that can be repurposed …

Discovery Of First-In-Class Small Molecule Agonists Of The Rxfp2 Receptor As Therapeutic Candidates For Osteoporosis, Maria Esteban Lopez

FIU Electronic Theses and Dissertations

Osteoporosis is a chronic bone disease characterized by decreased bone mass and increased risk of developing fractures, predominantly observed in the elderly. The pathophysiological cause of the disease is a decrease in the activity of the bone-forming cells (osteoblasts) that alters bone remodeling in favor of bone resorption, leading to a decrease in bone mass. Recent studies identified the relaxin family peptide receptor 2 (RXFP2), the G protein-coupled receptor (GPCR) for insulin-like 3 peptide (INSL3), as an attractive target expressed in osteoblast cells to increase bone formation. The goal of this dissertation is to discover and characterize small molecule agonists …

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 …

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 …

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 …

Evolutionary Expansions And Neofunctionalization Of Ionotropic Glutamate Receptors In Cnidaria, Ellen G. Dow

FIU Electronic Theses and Dissertations

Reef ecosystems are composed of a variety of organisms, transient species of fish and invertebrates, microscopic bacteria and viruses, and structural organisms that build the living foundation, coral. Sessile cnidarians, corals and anemones, interpret dynamic environments of organisms and abiotic factors through a molecular interface. Recognition of foreign molecules occurs through innate immunity via receptors identifying conserved molecular patterns. Similarly, chemosensory receptors monitor the environment through specific ligands. Chemosensory receptors include ionotropic glutamate receptors (iGluRs), transmembrane ion channels involved in chemical sensing and neural signal transduction. Recently, an iGluR homolog was implicated in cnidarian immunological resistance to recurrent infections of …

Computational Analysis Of Large-Scale Trends And Dynamics In Eukaryotic Protein Family Evolution, Joseph Boehm Ahrens

FIU Electronic Theses and Dissertations

The myriad protein-coding genes found in present-day eukaryotes arose from a combination of speciation and gene duplication events, spanning more than one billion years of evolution. Notably, as these proteins evolved, the individual residues at each site in their amino acid sequences were replaced at markedly different rates. The relationship between protein structure, protein function, and site-specific rates of amino acid replacement is a topic of ongoing research. Additionally, there is much interest in the different evolutionary constraints imposed on sequences related by speciation (orthologs) versus sequences related by gene duplication (paralogs). A principal aim of this dissertation is to …

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 …

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 …

Regulation Of Juvenile Hormone Synthesis By 20-Hydroxyecdysone In The Yellow-Fever Mosquito, Aedes Aegypti, Maria Areiza

FIU Electronic Theses and Dissertations

In Aedes aegypti, development and reproduction are regulated by juvenile hormone III (JH). This master regulatory hormone is synthesized by the corpora allata (CA), a pair of endocrine glands with neural connections to the brain. JH titers are largely determined by the rate of biosynthetic activity of the CA and are regulated by inhibitory and stimulatory factors. Like JH, the ecdysteroid 20-hydroxyecdysone (20E) is a key hormonal regulator and has been proposed as an allatoregulator in other insects. However, its part in the regulation of JH biosynthesis of mosquitoes was unknown. The specific aims of this dissertation were to …

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 Effect Of Target-Specific Biomolecules In Breast Cancer, Mohannad Garoub

FIU Electronic Theses and Dissertations

Cancer is the second leading cause of mortality in the United States and the World, therefore, early effective prevention, diagnosis, and therapy is needed. Estrogens play a major role in the initiation and progression of breast cancer. Elevated lifetime exposure to estrogens is associated with an increased risk of developing breast cancer. Estrogens through influencing mitochondria contribute to estrogen induced breast carcinogenesis; however, the exact mitochondrial mechanisms underlying the estrogen carcinogenic effect in breast tissue are not clearly understood. For this dissertation, the mitotoxic and cytotoxic effects of triphenylphosphonium cation (TPP) and Origanum majorana organic extract (OME) as well as …

Protein-Protein Interactions Of Bacterial Topoisomerase I, Srikanth Banda

FIU Electronic Theses and Dissertations

Protein-protein interactions (PPIs) are essential features of cellular processes including DNA replication, transcription, translation, recombination, and repair. In my study, the protein interactions of bacterial DNA topoisomerase I, an essential enzyme, were investigated. The topoisomerase I in bacteria relaxes excess negative supercoiling on DNA and maintains genomic stability. Investigating the PPI network of DNA topoisomerase I can further our understanding of the various functional roles of this enzyme. My study is focused on topoisomerase I of Escherichia coli and Mycobacterium smegmatis. Firstly, we have explored the biochemical mechanisms for an interaction between RNA Polymerase, and topoisomerase I in E. …

Biochemical Characterization Of Arsi: A Novel C-As Lyase For Degradation Of Environmental Organoarsenicals, Shashank Suryakant Pawitwar

FIU Electronic Theses and Dissertations

Organoarsenicals such as methylarsenical methylarsenate (MAs(V)) and aromatic arsenicals including roxarsone (4-hydroxy-3-nitrophenylarsenate or Rox(V)) have been extensively used as an herbicide and growth enhancers in animal husbandry, respectively. They undergo environmental degradation to more toxic inorganic arsenite (As(III)) that contaminates crops and drinking water. We previously identified a bacterial gene (arsI) responsible for aerobic MAs(III) demethylation. The gene product, ArsI, is a Fe(II)-dependent extradiol dioxygenase that cleaves the carbon-arsenic (C-As) bond in MAs(III) and trivalent aromatic arsenicals. The objective of this study was to elucidate the ArsI mechanism. Using isothermal titration calorimetry, we determined the dissociation constants (Kd) and ligand-to-protein …

Transcriptomic And Epigenetic Responses To Environmental Stress In Marine Bivalves With A Focus On Harmful Algal Blooms, Maria Victoria Suarez Ulloa

FIU Electronic Theses and Dissertations

Global change poses new threats for life in the oceans forcing marine organisms to respond through molecular acclimatory and adaptive strategies. Although bivalve molluscs are particularly tolerant and resilient to environmental stress, they must now face the challenge of more frequent and severe Harmful Algal Blooms (HABs) episodes. These massive outbreaks of microalgae produce toxins that accumulate in the tissues of these filter-feeder organisms, causing changes in their gene expression profiles, which in turn modify their phenotype in order to maintain homeostasis. Such modifications in gene expression are modulated by epigenetic mechanisms elicited by specific environmental stimuli, laying the foundations …

Screening Of Plants For Antibacterial Properties: Growth Inhibition Of Staphylococcus Aureus By Artemisia Tridentata, Steven Ross Eichelbaum

FIU Electronic Theses and Dissertations

Drug-resistant pathogenic and opportunistic bacteria are increasing in occurrence and prevalence, and pose a dangerous threat to human health. In the search for novel antibiotics with which to combat this threat, plants, specifically those used in traditional medicine with ascribed antibacterial properties, offer a promising and potentially vast source of such therapeutic compounds. The purpose of this study was therefore to screen chemical extracts created from various plant species for antibacterial properties versus pathogenic bacterial species. In the course of these antibacterial assays, we successfully identified a methanol extract derived from Artemisia tridentata tridentata plant material as capable of inhibiting …

Microbial Functional Diversity And The Associated Biogeochemical Interactions Across Miami-Dade County, Florida Soils, Priyanka Kushwaha

FIU Electronic Theses and Dissertations

Decomposition of soil organic matter by microbial processes results in carbon sequestration within soils and/or carbon loss via atmospheric emission of carbon dioxide and methane. Natural as well as anthropogenic factors have been documented to impact soil microbial diversity and the associated biogeochemical functions. The soil microbial communities co-inhabiting Miami-Dade County soils, Florida are under threat because of the ongoing restoration efforts in the adjoining Florida Everglades Parks, predicted climatic changes such as sea-level rise and high rainfall, as well as urbanization. Therefore, an improved understanding of the current microbial functional communities is essential to better assess the impact of …

Regulation Of Particle Uptake By Pp2a/B56 And Lkb1 In Dictyostelium Discoideum, Mujataba Rahiman Sharief

FIU Electronic Theses and Dissertations

Dictyostelium discoideum is a soil dwelling amoeba which has been widely used as a model organism to study cellular processes such as signal transduction, chemotaxis, endocytosis and exocytosis. The process of phagocytosis in Dicytostelium is largely comparable to that of neutrophils and macrophages in the mammalian system. Neutrophils and macrophages are cells of the innate immune system and they engulf infectious bacteria through phagocytosis. Dictyostelium cells uptake yeast and bacteria for their nutrition through phagocytosis, which is an actin dependent mechanism and is a target of multiple signaling inputs. Recent studies have uncovered different proteins involved in the signaling of …

Conformational Dynamics And Stability Associated With Magnesium Or Calcium Binding To Dream In The Regulation Of Interactions Between Dream And Dna Or Presenilins, Khoa Ngoc Pham

FIU Electronic Theses and Dissertations

Downstream regulatory element antagonist modulator (DREAM) is involved in various interactions with targets both inside and outside of the nucleus. In the cytoplasm, DREAM interacts with the C-terminal fragments of presenilins to facilitate the production of β-amyloid plaques in Alzheimer’s disease. In the nucleus, Ca²⁺ free DREAM directly binds to specific downstream regulatory elements of prodynorphin/c-fos gene to repress the gene transcription in pain modulation. These interactions are regulated by Ca²⁺ and/or Mg²⁺ association at the EF-hands in DREAM. Therefore, understanding the conformational dynamics and stability associated with Ca²⁺ and/or Mg²⁺ binding to DREAM …

Protein-Ligand Interactions And Allosteric Regulation Of Activity In Dream Protein, Walter G. Gonzalez

FIU Electronic Theses and Dissertations

Downstream regulatory antagonist modulator (DREAM) is a calcium sensing protein that co-assembles with K_V4 potassium channels to regulate ion currents as well as with DNA in the nucleus, where it regulates gene expression. The interaction of DREAM with A-type K_V4 channels and DNA has been shown to regulate neuronal signaling, pain sensing, and memory retention. The role of DREAM in modulation of pain, onset of Alzheimer’s disease, and cardiac pacemaking has set this protein as a novel therapeutic target. Moreover, previous results have shown a Ca²⁺ dependent interaction between DREAM and K_V4/DNA involving …

Characterization Of An Epoxide Hydrolase From The Florida Red Tide Dinoflagellate, Karenia Brevis, Pengfei Sun

FIU Electronic Theses and Dissertations

Polyether compounds are a subgroup of natural products with regular occurrence of multiple C-O-C motifs. The biosynthetic origin of polycylic polethers has been studied and the majority of them are derived from polyketide or terpene pathways. Normally, the polycyclic polyethers can be divided into two groups based on their structural features: the first group features multiple rings that are interconnected by carbon-carbon single bond, which are produced by a biosynthetic cascade of exo epoxide-opening reactions; the other group has multiple fused cyclic ethers and are formed by an cascade of endo epoxide-opening reactions.

Karenia brevis (K. brevis) is …

The Effect Of Sample And Sample Matrix On Dna Processing: Mechanisms For The Detection And Management Of Inhibition In Forensic Samples, Lilliana I. Moreno

FIU Electronic Theses and Dissertations

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments …

Redox Regulation Of Ras Proteins In Dictyostelium Discoideum, Boris Castillo Chabeco

FIU Electronic Theses and Dissertations

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide.

The main objective of this project was to determine the mechanism of …

Multimode Analysis Of Nanoscale Biomolecular Interactions, Purushottam Babu Tiwari

FIU Electronic Theses and Dissertations

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical …