Biochemistry Commons^™

Binding Interactions Of Biologically Relevant Molecules Studied Using Surface-Modified And Nanostructured Surfaces, Palak Sondhi

Dissertations

This research focuses on the field of surface nanobioscience, wherein different nanosurfaces that will be used as working electrodes in the electrochemical cell are manufactured and surface modified to understand the critical binding interactions between biologically significant molecules like proteins, carbohydrates, small drug molecules, and glycoproteins. This research is essential if we are to determine whether a synthetic molecule can serve as a therapeutic candidate or diagnose a disease in its early stages. In order to fully understand the binding interactions, the study begins with defining some of the fundamental concepts, principles, and analytical tools for biosensing.

Afterwards, we addressed …

Canagliflozin Ameliorates Autistic-Like Features And Mitigates Brain Oxidative Stress Levels In Valproic Acid-Induced Autism In Rats, Mohammad Moutaz Nakhal

Theses

Autism spectrum disorder (ASD) is a neurodevelopmental disease with a substantially increasing incidence rate. It is mainly characterized by repetitive behavior, intellectual difficulties, social communication and interactions deficits. Many medications, dietary supplements, and behavioral treatments have been recommended for ASD management, however, there is no cure yet. Recent studies have examined the therapeutic potential of the sodium-glucose cotransporter 2 (SGLT2) inhibitors in neurodevelopmental diseases, based on their proved anti-inflammatory and antioxidant effects. The main objective of this study is to assess the ability of canagliflozin in improving the behavioural characteristics of autistic rats and investigate the efficacy of canagliflozin in …

Acetylation Regulates Thioredoxin Reductase Activity And Oligomerization, David E. Wright

Electronic Thesis and Dissertation Repository

The Thioredoxin (Trx) system provides the cell with robust defense against oxidative stress and regulates the function of nearly every cellular process through the reduction-oxidation (redox) regulation of proteins. The Trx system is involved in the development of many diseases ranging from cancer to cardiovascular disorders. Thioredoxin reductase (TrxR) is the key enzyme in the Trx system and contains the 21^st genetically encoded amino acid, selenocysteine (Sec). There were multiple experimentally identified TrxR acetylation sites with an unknown effect on TrxR activity. My thesis tested the hypothesis that programmed protein acetylation will enhance the activity of TrxR1. I used …

Short-Term Versus Long-Term Effects Of Nitrogen Addition And Warming On Ecosystem N Dynamics In A Grass-Dominated Temperate Old Field, Benjamin F. A. Souriol

Electronic Thesis and Dissertation Repository

Increased atmospheric nitrogen (N) deposition and climate warming are both anticipated to influence the ecosystem N dynamics of northern temperate ecosystems substantially over the next century. Nevertheless, in field experiments with N addition and warming treatments, temporal scale can play an important role in determining the extent of treatment effects on N dynamics, and it is unclear to what extent the results of short-term studies can be extrapolated to responses over longer time scales. I compared the short-term versus long-term effects of N addition and warming on net N mineralization, N leaching, and N retention in a grass-dominated old field. …

Structural Insights Into The Cl-Par-4 Protein: Ionic Requirements, Conformational Transitions, And Interaction With Cisplatin, Krishna Kumar Raut

Chemistry & Biochemistry Theses & Dissertations

Cancer continues to be the leading global cause of death, with challenges in early diagnosis, drug resistance, non-specific drug targeting, and cancer recurrence and metastasis posing formidable obstacles in cancer therapy. In this context, Prostate Apoptosis Response-4 (Par-4), a pro-apoptotic tumor suppressor protein, emerged as a promising therapeutic target due to its ability to selectively induce apoptosis in cancer cells, thereby minimizing the drug-associated adverse effects. However, a comprehensive understanding of the structural features of Par-4, specifically the caspase-cleaved fragment (cl-Par-4), is crucial for therapeutic advancements.

This dissertation investigated the effects of various ions, both monovalent and divalent, on the …

A Tale Of Two Mechanisms: Is The P53 Modulator Coti-2 A Zinc Chaperone Instead?, Irem Simsek

Electronic Theses and Dissertations

The tumor protein p53 plays a vital role in regulating protein pathways that determine the fate of cells. Any interference with p53 function can cause disruptive cell proliferation and carcinogenesis. The p53 protein experiences a high frequency of mutation in human cancers, leading to significant research efforts to target mutant p53 and restore the normal functioning of p53. The COTI-2 small molecule, developed by COTINGA Pharmaceuticals through a computational program, has entered Phase I clinical trials. Although the mechanism of action is not fully understood, it has shown considerable promise in targeting cell lines with mutant p53. COTI-2 is believed …

Characterization Of Dna Regulatory Elements Of The Highly Abundant Secreted Protein 1 (Hasp1) Promoter And Analysis Of Transcriptomic Responses Under Phosphate Depletion In The Diatom Phaeodactylum Tricornutum, Mahsa Farmanbar

Electronic Thesis and Dissertation Repository

Phosphorus (P) is essential for all life. The bioavailability of phosphorus in oceans impacts diatoms like Phaeodactylum tricornutum (P. tricornutum). P. tricornutum is extensively used in transcriptomic studies to understand pathways involved in P-acquisition. However, activation mechanisms and roles of regulatory elements in P-acquisition responses remains unclear. Here, I deleted predicted phosphate regulatory sites in P.tricornutum’s HASP1 promoter to create different HASP1-eGFP constructs. Under P-depletion, two constructs showed increased eGFP secretion. Additionally, a HASP1 knockout strain was grown under different phosphorus sources to determine if HASP1 is a phytase. Cells grown in full phosphorus utilized organic …

Protein Stability In Solution And In The Gas Phase., Yousef Haidar

Electronic Thesis and Dissertation Repository

Electrospray Ionization mass spectrometry (ESI-MS) is widely used for probing proteins, yet many aspects of this technique remain elusive. Using MS, ion mobility spectrometry (IMS), and circular dichroism (CD) spectroscopy, this thesis sheds light on the stability differences of proteins in the gas phase and solution. After a general introduction (Chapter 1), Chapter 2 scrutinizes some aspects of native ESI. Our data highlight the significance of cone voltage in maintaining a native-like fold and show the advantage of using NH₄Ac in protein experiments. Chapter 3 focuses on hydrogen/deuterium exchange (HDX)-MS. Several studies have reported that D₂O …

Development Of Antiviral Peptidomimetics, Songyi Xue

USF Tampa Graduate Theses and Dissertations

Protein-protein interactions (PPIs) are essential for biological processes and are associated with a number of diseases, including cancer, infectious diseases, and neurodegenerative diseases. As a result, modulation of PPIs has been recognized as one of the most promising strategies to develop the novel drugs. Peptide modulators always exhibit higher specificity and affinities with targets than small compounds or monoclonal antibodies, but their broad medicinal effectiveness is constrained by their poor bioavailability and biostability. Peptidomimetics, which have been developed to mimic the structure as well as function of bioactive peptides and proteins, have shown excellent potential in protein surface mimicry and …

Characterization Of Pathological Tau Mutants, Charles J. Mcdonald

Dissertations, Theses, and Capstone Projects

Tau is a protein expressed exclusively in glia and neurons in the central nervous system and implicated in several neurogenerative diseases called “tauopathies”. Among all the tauopathies, one third is characterized by the presence of genetic mutations leading to the synthesis of tau proteins with single amino acid substitutions at specific locations and affecting protein function. While most of the initial studies have emphasize the functional role of tau as modulator of the axonal cytoskeleton, it has recently been well accepted that tau is also an intrinsically disordered protein that tends to form membraneless organelles called coacervates, due to a …

Magnetically-Driven Therapeutic Agents Delivery System Using Iron Oxide Nanocages And Enhancement Of Exosome Secretion, A Potential Biological Drug Delivery Carrier, Min A Kang

Dissertations, Theses, and Capstone Projects

Nano-scale particles have attracted research attention due to their differences in properties such as penetration, circulation, and toxicity compared to bulk materials. This thesis mainly focused on using 20 nm iron oxide nanoparticles as siRNA delivery carriers under the alternating magnetic field and the development of a method to amplify the secretion of 150 nm exosomes from the cells, which could potentially use as a biological drug carrier.

Chapter 2 discusses a magnetically driven nanoparticle therapeutic agent delivery system, which efficiently modified the gene expression post-transcriptionally. In this work, we examined whether the caged-shaped 20nm iron oxide nanoparticles (IO-nanocages) can …

Construction And Performance Optimization Of Bioconjugated Nanosensors For Early Detection Of Breast Cancer And Pro-Inflammatory Diseases, Pooja Gaikwad

Dissertations, Theses, and Capstone Projects

In recent years, nanosensors have emerged as a tool with strong potential in medical diagnostics. Single-walled carbon nanotube (SWCNT) based optical nanosensors have notably garnered interest due to the unique characteristics of their near-infrared fluorescence emission, including tissue transparency, photostability, and various chiralities with discrete absorption and fluorescence emission bands. Additionally, the optoelectronic properties of SWCNT are sensitive to the surrounding environment, which makes them suitable for in vitro and in vivo biosensing. Single-stranded (ss) DNA-wrapped SWCNTs have been reported as optical nanosensors for cancers and metabolic diseases. Breast cancer and cardiovascular diseases are the most common causes of death …

Characterization Of The Conformational Binding Of N-Methyl Mesoporphyrin Ix With Dna Model Telomeric G-Quadruplex Forming Sequences, Jessica Desamero

Dissertations, Theses, and Capstone Projects

DNA G-quadruplexes are alternative secondary structures formed from guanine rich DNA sequences. G-quadruplexes are found most abundantly at the ends of chromosomes called telomeres, where they can ultimately prevent further tumor growth and progression. Thus, quadruplex interactive agents, or ligands that can bind to and stabilize DNA G-quadruplex structures, are of high interest for their potential chemotherapeutic abilities. One quadruplex interactive agent is the porphyrin N-methyl mesoporphyrin IX (NMM), which has high binding selectivity for quadruplex structures. While the NMM-quadruplex complex in crystallized form has been greatly studied, this complex in solution form is not as well studied. Through circular …

Nanodiscs: A Novel Approach To The Study Of The Methionine Abc Transporter System, Michael T. Winslow

Master's Theses

Membrane transporter proteins play the vital role of moving compounds in and out of the cell and are essential for all living organisms. ATP Binding Cassette (ABC) family transporters function both as importers and exporters in prokaryotes. MetNI is an E. coli Type I ABC transporter responsible for the uptake of methionine into the cytosol from the cell periplasmic space through the cell membrane to maintain intracellular methionine pools. ABC transporters, like other membrane proteins, are most often mechanistically and structurally studied in vitro, solubilized by detergents. However, detergent micelles may affect the conformational changes of membrane proteins relative to …

State-Of-The-Art Approaches For Sequencing, Assembling And Annotating Naphthenic Acid Degrading Bacterial Metagenomes, Henry H. Say

Electronic Thesis and Dissertation Repository

Naphthenic acids (NAs) are the main toxic component of oil refinery wastewater and require special processes to be removed. Harnessing bacterial biodegradation for NA removal has the potential to be effective, yet NA-degrading bacteria and pathways are poorly understood and uncharacterized. To improve our understanding of NA degradation, I characterize the metagenomes of novel NA-degrading bacterial communities seeded in NA-enriched granulated activated carbon (GAC) filters. I demonstrate methods that maximize the throughput of extraction, sequencing, and annotation of novel metagenomes - producing 72 MAGs and other 5432 circular contigs - 226 of which were putative phages. I also include state-of-the-art …

The Effects Of Resistance Exercise Training On Insulin Resistance Development In Female Rodents With Type 1 Diabetes, Mitchell James Sammut

Electronic Thesis and Dissertation Repository

The etiology of insulin resistance (IR) development in type 1 diabetes mellitus (T1DM) remains unclear; however, impaired skeletal muscle metabolism may play a role. While IR development has been established in male T1DM rodents, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1DM compared to aerobic exercise. Additionally, the molecular mechanisms mediating RT-induced improvements in insulin sensitivity remain unclear. Therefore, the purpose of this study was to investigate the effects of RT on IR development in female …

Upland Cotton And Nematodes: An Analysis Of Historical Resistance, Upcoming Threats, And Co-Inoculation Effects, Amanda Gaudin

Theses and Dissertations

Upland cotton (Gossypium hirsutum ) is an important fiber crop grown throughout the southern United States. Plant-pathogenic nematodes are worm-like animals that feed on the roots of most agronomic crops, including cotton. The southern root-knot nematode (Meloidogyne incognita, RKN) and the reniform nematode (Rotylenchulus reniformis, RN) cause significant yield losses in cotton every year. Current sources of resistance are effective but limited, therefore historical screenings of cotton accessions were revisited in search for novel resistance sources. None were identified but many of the screened accessions possessed markers of known root-knot nematode and reniform nematode resistance. …

Selective Activation Of Thrombin Activatable Fibrinolysis Inhibitor (Tafi) Attenuates Metastatic And Angiogenic Capabilities Of Melanoma And Lung Carcinoma In Vitro, Jacklyn Krizsan

Electronic Thesis and Dissertation Repository

Metastasis and angiogenesis are hallmarks of aggressive cancers, both depending on degradation of extracellular matrix by proteases such as plasmin. Plasmin activation is inhibited by thrombin-activatable fibrinolysis inhibitor (TAFI)-mediated cleavage of terminal lysine residues on plasminogen receptors. Activation of TAFI is most effectively done in complex with thrombomodulin (TM). TM is known to have anti-cancer properties, but it is not known if this is due to TAFI activation or an alternative substrate protein C (PC). We hypothesize that specific promotion of TAFI activation with TM treatment will attenuate metastatic and angiogenic capabilities of tumour cells.

Melanoma and lung carcinoma cells …

Fatty Acids And Parasitism: Towards A Better Understanding Of Lipid Metabolism In Trypanosoma Brucei, Joshua Saliutama

All Dissertations

Trypanosoma brucei is an extracellular eukaryotic parasite that causes sleeping sickness in humans and cattle. As an extracellular parasite, T. brucei relies on the host’s nutrients to satisfy its growth requirements. The parasite is unusual because it does not uptake most of the host’s lipid species. Instead, T. brucei prefers to perform de novo synthesis of most lipid species. One of the lipid species that T. brucei can both uptake and synthesize is fatty acids. In my thesis work, I investigated the dynamics of fatty acid uptake, metabolism, and utilization of T. brucei. My work starts by determining the …

X-Ray Crystal Structure Of E399q,E708q Ecm16 Double Mutant, Gileydis Guillama

Open Access Theses & Dissertations

The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats. However, their widespread utilization has contributed to the emergence and spread of antibiotic resistance, which now poses a significant public health challenge. Streptomyces bacterium, produce diverse secondary metabolites with antibacterial, antifungal, antiviral, antitumoral, and immunosuppressant activities. Among these compounds is echinomycin, a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi, which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps. A gene called ecm16 was identified in the echinomycin biosynthetic gene cluster, which provides echinomycin self-resistance. Ecm16 recognizes DNA duplexes …

Structural Study Of Lsd13 And Lsd14 Type I Modular Polyketide Synthases From The Lasalocid A Biosynthesis Pathway, Dayan Viera

Open Access Theses & Dissertations

Bioactive natural products often possess a complex structure and have a large size which make their chemical synthesis highly challenging. To overcome this challenge, we are developing a biosynthetic method for production of natural products and their analogs. This technology is expected to decrease the synthesis time from years to weeks and reduce the cost of drug production by multiple orders of magnitude. We are investigating the biosynthesis pathway of lasalocid A, an anticancer antibiotic, to develop this technology. Specifically, we aim to characterize the enzymes structurally and biochemically in this pathway, thus paving the way for their rational engineering. …

Determining The Existence And Regulation Of Microlipophagy In Primary Brown And White Adipocytes, Sara C. Arenas De Leon

Biomedical Sciences ETDs

Lipids are one of life’s four main macromolecules and provide essential functions to cells.

The degradation of lipid droplets and mobilization of lipids occurs through lipolysis. Emerging evidence demonstrates evidence of a selective form of autophagy in lipolysis. The process of microlipophagy has only recently been described, and many features of its regulation are still poorly understood. Our recent study showed that inhibiting mammalian target of rapamycin complex 1 (mTORC1) pathway by depletion of Raptor in adipocytes led to an influx of lysosomes and accumulation of lipid droplets within lysosome. Here, we expand on these previous findings. We were able …

Phenotypic And Transcriptomic Characterization Of Rice Snrk1 Mutants Developed By Crispr/Cas9 Mutagenesis, Maria Clara Faria Chaves

Graduate Theses and Dissertations

SnRK1 is a heterotrimeric protein kinase that is composed of a catalytic subunit (α) and two regulatory subunits (β and βγ), and it has a main role in regulating energy homeostasis in the plant by modulating anabolic and catabolic process. SnRK1 phosphorylates and alters the activities of enzymes involved in metabolism and regulates gene expression by altering the activity of chromatin-remodeling enzymes or the transcription factors. Rice contains three functional paralogs of SnRK1α: SnRK1αa (LOC_Os03g17980), SnRK1αb (LOC_Os08g37800), and SnRK1αc (LOC_Os05g45420). This study focused on the function of these SnRK1 paralogs by evaluating the phenotypic and transcriptomic characteristics and the disease …

Epitranscriptomic Regulation In Breast Cancer And Pcb-Induced Liver Disease., Belinda Petri

Electronic Theses and Dissertations

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, …

Fbg Αc 389 – 402 Modulates Factor Xiii Crosslinking In The Fibrinogen Αc Region., Francis Dean Orlina Ablan

Electronic Theses and Dissertations

Fibrinogen (Fbg) is a coagulation protein critical for clot formation. Coagulation Factor XIII (FXIII) is a calcium-dependent transglutaminase that crosslinks reactive glutamines (Q) and lysines (K) between fibrin and other anti-fibrinolytic proteins. In the presence of Ca²⁺, FXIII could be activated non-proteolytically (FXIII-A°), or proteolytically by thrombin (FXIII-A*). Significant increases in clot stability and red blood cell retention are linked to FXIII activity in the fibrinogen αC region (Fbg Aα 221 – 610). This region contains several FXIII-reactive glutamines and lysines, as well as a binding site for FXIII-A* (Fbg αC 389 – 402) that includes a key …

The Inner Workings Of Thrombin-Activatable Fibrinolysis Inhibitor: A Study Of The Tm-Mediated Activation Of Tafi And Inactivation Of Tafia, Haley D N Marier

Electronic Thesis and Dissertation Repository

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a key regulator of hemostasis and inflammation. TAFI is activated by thrombin proteolysis, most effectively when in complex with cofactor thrombomodulin (TM). Soluble TM, encompassing EGF-like domains 3 through 6 is sufficient to promote TAFI activation, though the role of EGF 4 has not been assessed. Through rearrangement of the soluble TM domains, we determined that EGF 4 was acting as a spacer to promote TAFI activation. Activated TAFI (TAFIa) is a metastable enzyme, with a half-life of only 8-15 min at 37^oC. The mobile loop of TAFIa was expected to modulate stability; …

Uvrd1 Helicase Of Mycobacterium Tuberculosis That Can Be Activated By Multiple Unique Mechanisms, Ankita Chadda

Arts & Sciences Electronic Theses and Dissertations

Abstract Mycobacterium tuberculosis (Mtb) causes tuberculosis disease in humans and is one of the leading causes of death worldwide due to infectious agents. During infection, Mtb is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that causes DNA damage. UvrD-like helicases are involved in DNA repair and use energy from ATP hydrolysis to translocate on single stranded DNA (ssDNA) or unwind double-stranded DNA (dsDNA) to remove the damaged DNA strand. Previous studies on UvrD-like helicases have shown that they exist in a monomer-dimer equilibrium and unwind only as dimers in the absence of accessory …

Advances In Phaeodactylum Tricornutum Nuclear Engineering, Mark Pampuch

Electronic Thesis and Dissertation Repository

The marine diatom Phaeodactylum tricornutum has the potential to become an excellent platform for the sustainable production of valuable compounds and pharmaceuticals, but currently large-scale engineering of this organism remains a challenge due factors like inefficient genetic transformation protocols and a lack of accurate genomic data. This thesis addresses these two bottlenecks by (i) optimizing an electroporation protocol to P. tricornutum and (ii) remapping genomic data from a scaffolded genome assembly to a telomere-to-telomere genome assembly. An optimized transformation protocol was developed that could consistently transform blunt-ended and DNA with overhangs and yielded up to 1000+ colony forming units per …

Elucidating The Biomechanics Of Mertk-Mediated Efferocytosis, Brandon Hayato Dickson

Electronic Thesis and Dissertation Repository

Macrophages are key mediators of efferocytosis – the phagocytic engulfment and removal of apoptotic cells. During engulfment, the coordinated activity of efferocytic receptors induces the remodeling of the actin cytoskeleton, which facilitates the envelopment of the cell by the plasma membrane. Mer receptor tyrosine kinase (MERTK) is a crucial efferocytic receptor, but its role during actin remodeling is not well understood. Previously, our lab showed that MERTK is an activator of β₂ integrins – which are comprised of receptors known to induce the actin polymerization that is required for engulfment. We hypothesized that MERTK is an indirect stimulator of …

Functionalizing Conjugative Systems To Deliver Crispr Nucleases For Targeted Bacterial Killing, Thomas A. Hamilton

Electronic Thesis and Dissertation Repository

The interactions between humans and microbes are intimately important to human health, with both commensal and pathogenic bacteria affecting homeostasis and disease. Increasing concern over antibiotic resistance in bacterial pathogens represents a significant threat to human health, and use of traditional antibiotics to treat infections can be detrimental to commensal bacteria as well as pathogens, demonstrating a need for more specific antibacterial reagents. RNA-guided CRISPR nucleases, which can target and cleave genomes of interest, are a potential tool for specific bacterial targeting. A key limitation to the use of CRISPR antimicrobials is effective and robust delivery to the target bacteria. …