Biochemistry, Biophysics, and Structural Biology Commons^™

Insights Into The Therapeutic Potential Of Salt Inducible Kinase 1: A Novel Mechanism Of Metabolic Control, Randi Fitzgibbon

Dissertations and Theses (Open Access)

Salt inducible kinase 1 (SIK1) has been considered a stress-inducible kinase since it was first cloned in 1999. Continued efforts since this time have been dedicated to characterizing the structure and function of SIK1. Such research has laid the ground work for our understanding of SIK1 action and regulation in tissue and stimuli dependent manners. The fundamental findings of this dissertation continue in this tradition and include investigations of SIK1 regulatory mechanisms in skeletal muscle cells, the cellular and physiological effects of SIK1 loss of function in vitro and in vivo, and intracellular metabolic and mitochondrial regulation by this …

Endocytic Trafficking Of The Amyloid Precursor Protein In Rat Cortical Neurons, Sahily Reyes

Dissertations and Theses (Open Access)

Amyloid-beta (Aβ) aggregation and deposition into extracellular plaques is a hallmark of the most common forms of dementia, including Alzheimer’s disease. The Aβ-containing plaques result from pathogenic cleavage of amyloid precursor protein (APP) by secretases resulting in intracellular production of Aβ peptides that are secreted and accumulate extracellularly. Despite considerable progress towards understanding APP processing and Aβ aggregation, the mechanisms underlying endosomal production of Aβ peptides and their secretion remain unclear. Using endosomes isolated from cultured primary neurons, we determined that the trafficking of APP from the endosomal membrane into internal vesicles of late endosome/multivesicular bodies (MVB) is dependent on …

Cellular Mechanisms Of Ionoregulation In The Gill Of Japanese Medaka And Rainbow Trout, Rebecca Jo Bollinger

Graduate Theses and Dissertations

Euryhaline fishes are capable of adapting to a wide range of salinities such as freshwater, brackish water or seawater. Through the combined effort of the gill, kidney and intestine, they are able to osmoregulate to maintain a constant internal hydromineral balance. As the gill is in direct contact with the external environment, it is continuously working to maintain ion and acid/base balance, gas exchange and eliminate nitrogenous waste. Fish in freshwater are subjected to osmotic water gain and diffusional ion loss across the gill and experience the opposite in seawater. Therefore, the gill exhibits extreme plasticity when experiencing a change …

Determination Of The Effects Of Different Maillard Reaction Products On The Taxonomic Composition Of The Gut Microbiota, Nesreen Hamdan Aljahdali

Graduate Theses and Dissertations

The Maillard Reaction (MR) is a non-enzymatic chemical reaction which results in linkage between the amino group of amino acids and the carbonyl group of reduced sugars. This reaction generates Maillard reaction products (MRPs) which are not present naturally in foods, and are responsible for a range of colors, odors, flavors, and other sensory properties. Conflicting reports of MRPs impacts on human health are probably due to the fact that bioconversion of these digestible molecules by the gut microbiota has been marginally taken into account. This study aimed to determine the effects of different MRPs on rodent’s gut microbiota through16S …

Detection Of Survival And Proliferation Of Sulfate Reducers Under Simulated Martian Atmospheric And Soil Conditions, Sergio Mosquera Mora

Graduate Theses and Dissertations

Numerous studies have tried to determine the survivability and proliferation of microorganisms under simulated Martian conditions. Furthermore, most of them have been focused on the ability of these microbes to cope with high brines’ salt (NaCl) concentrations inherent of the Martian surface. However, there are not studies related to the ability of bacteria to survive on subsurface environments that have increasing concentrations of sulfate compounds. For this research, a group of microorganisms known as sulfate-reducing bacteria or simply sulfate reducers were chosen due to their ability to use sulfate compounds as terminal electron acceptors to produce metabolic energy, their tolerance …

Role Of Heat Shock Transcription Factor 1 In Ovarian Cancer Epithelial-Mesenchymal Transition And Drug Sensitivity, Chase David Powell

USF Tampa Graduate Theses and Dissertations

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned …

The Role Of Sgs1 And Exo1 In The Maintenance Of Genome Stability., Lillian Campos-Doerfler

USF Tampa Graduate Theses and Dissertations

Genome instability is a hallmark of human cancers. Patients with Bloom’s syndrome, a rare chromosome breakage syndrome caused by inactivation of the RecQ helicase BLM, result in phenotypes associated with accelerated aging and develop cancer at a very young age. Patients with Bloom’s syndrome exhibit hyper-recombination, but the role of BLM and increased genomic instability is not fully characterized. Sgs1, the only member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is known to act both in early and late stages of homology-dependent repair of DNA damage. Exo1, a 5′–3′ exonuclease, first discovered to play a role …

Endonucleolytic Cleavage In The Expansion Segment 7 Of 25s Rrna Is An Early Marker Of Low-Level Oxidative Stress In Yeast, Daniel Shedlovskiy, Jessica A Zinskie, Ethan Gardner, Dimitri G Pestov, Natalia Shcherbik

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of …

Association Of Vitamin D Deficiency And Vdbp Gene Polymorphism With The Risk Of Ami In A Pakistani Population, Mujtaba Mubashir, Shaheena Anwar, Asal Khan Tareen, Naseema Mehboobali, Khalida Iqbal, Mohammad Iqbal

Department of Biological & Biomedical Sciences

OBJECTIVE: To investigate the relationship of vitamin D deficiency and risk of AMI in a Pakistani population, and to find out any associationbetween vitamin D binding protein (VDBP) genotypes and risk of AMI in this population.
METHODS: In a comparative cross-sectional study, 246 patients (age: 20-70 years; 171 males and 75 females) with first AMI were enrolled with informed consent. Similarly, 345 healthy adults (230 males and 115 females) were enrolled as controls. Their fasting serum samples were analyzed for 25 (OH) vitamin D, lipids and other biomarkers using kit methods, while DNA was analyzed for VDBP genotypes using PCR-RFLP …

Nanoparticle Delivery Of Mir-34a Eradicates Long-Term-Cultured Breast Cancer Stem Cells Via Targeting C22orf28 Directly, Xiaoti Lin, Weiyu Chen, Fengqin Wei, Binhua P. Zhou, Mien-Chie Hung, Xiaoming Xie

Molecular and Cellular Biochemistry Faculty Publications

Rationale: Cancer stem cells (CSCs) have been implicated as the seeds of therapeutic resistance and metastasis, due to their unique abilities of self-renew, wide differentiation potentials and resistance to most conventional therapies. It is a proactive strategy for cancer therapy to eradicate CSCs. Methods: Tumor tissue-derived breast CSCs (BCSC), including XM322 and XM607, were isolated by fluorescence-activated cell sorting (FACS); while cell line-derived BCSC, including MDA-MB-231.SC and MCF-7.SC, were purified by magnetic-activated cell sorting (MACS). Analyses of microRNA and mRNA expression array profiles were performed in multiple breast cell lines. The mentioned nanoparticles were constructed following the standard molecular cloning …

Characterization Of Different Molecular Markers For Identification Of Salmonella Enterica Serovar Typhi In Pakistani Population, Faizan Muttiullah, Fida Muhammad Khan, Fakhar-I- Abbas, Sabiha Shamim

Journal of Bioresource Management

Typhoid is caused by Salmonella enterica serovar Typhi that is usually diagnosed by using serologic and immuno-chromatographic techniques in developing counties including Pakistan, which is thought to be an unreliable diagnostic method. For accurate diagnosis we used molecular techniques to amplify 204 bp StyR-36 and 498 bp flagellin gene for the identification of Salmonella enterica serovar Typhi. This study was done on 58 individuals diagnosed positive of typhoid via serologic tests and 50 healthy individuals as a control group. Success rate of amplification for flagellin gene was 77.58% while that for StyR-36 gene was 68.97% showing that flagellin gene primer …

Exploring Biological Heterogeneity And Its Consequences At Tissue And Cellular Scales Through Mathematical And Computational Modeling, Romica Kerketta

Biomedical Sciences ETDs

This dissertation explores the effects of heterogeneity across different biological scales in cancer as well as normal cells. At the tissue scale, we investigated the variability present in the tumor microenvironment and its effect on patient chemotherapeutic outcomes using a mathematical model of drug transport. We found that parameters such as tumor blood perfusion and radius of blood vessel had an impact on the tumor cytotoxicity. This indicated that the physical microenvironment of the tumor is an important regulator of the tumor response to chemotherapy. At the cellular scale, we investigated the heterogeneity present on the membrane landscape of ErbB2 …

Stabilization Of The Transcription Factors Slug And Twist By The Deubiquitinase Dub3 Is A Key Requirement For Tumor Metastasis, Yiwei Lin, Yu Wang, Qing Shi, Qian Yu, Cuicui Liu, Jing Feng, Jiong Deng, B. Mark Evers, Binhua P. Zhou, Yadi Wu

Molecular and Cellular Biochemistry Faculty Publications

The Epithelial-mesenchymal transition (EMT) represents a cellular de-differentiation process that provides cells with the increased plasticity required during embryonic development, tissue remodeling, wound healing and metastasis. Slug and Twist are two key EMT transcription factors (EMT-TFs) that are tightly regulated via ubiquitination and degradation. How Slug and Twist escape degradation and become stabilized in cancer cells remains unclear. One plausible mechanism of Slug and Twist stabilization involves removal of ubiquitin by deubiquitinases (DUBs). In this study, we identified Dub3 as a novel DUB for both Slug and Twist. We further found that Dub3 overexpression increased Slug and Twist protein levels …

Molecular Mechanisms Of C-Terminal Eps15 Homology Domain Containing (Ehd) Protein Function, Kriti Bahl

Theses & Dissertations

Endocytic trafficking is not only an essential process for the maintenance of cellular homeostasis but also plays a vital role in regulating diverse cellular processes such as signaling, migration and cell division. The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play pivotal roles in regulating distinct steps of endocytic trafficking. Among the EHDs, EHD2 is disparate both in terms of sequence homology (70%) and its subcellular localization at the caveolae. The crystal structure of EHD2 has been solved and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. However, the other paralogs EHD1, …

Molecular Mechanisms Of Dna Replication Initiation In Hpvs With Genetic Variations Leading To Cellular Carcinogenesis, Gulden Yilmaz

Graduate School of Biomedical Sciences Theses and Dissertations

Human papillomaviruses are a vast family of double-stranded DNA viruses containing non-carcinogenic and carcinogenic types, whose crucial differences remain unknown, except for the difference in the frequency of DNA replication. The human papillomavirus (HPV) E2 protein regulates the initiation of viral DNA replication and transcription. Its recognition and binding to four 12 bp palindromic sequences in the viral origin is essential for its function. Little is known about the DNA binding mechanism of the E2 protein found in HPV types that have low risk for oncogenicity (low-risk) as well as the roles of various elements of the individual binding sites. …

Chaperoning Ef Hands That Shape Calcium Response: Ncald, Hpca And S100b, Jingyi Zhang

Graduate School of Biomedical Sciences Theses and Dissertations

All organisms have an internal clock with a defined period between repetitions of activities. The period for circadian clock in human is 24.5 hours, while in mouse and rat, it is 23.5 hours. However, all organisms are forced to be in synchronization with their environment. A major environmental force that resets the internal clock to 24 hours is light. This phenomenon is defined as “light entrainment” or “phase-setting”. It is unclear how this entrainment process occurs. Studies from this laboratory indicate a role for two neuronal calcium sensor proteins: Neurocalcin  (NCALD) and S100B. For these two genes, mRNA as …

Linear Dna-Linked Nanoparticle Building Blocks (Nblocks) For Modular Self-Assembly Of Nanostructures, Jakob Thomas Hockman

Graduate Theses and Dissertations

Controlling the shapes and sizes of nanomaterials often enables controlling their properties for certain applications. The most promising methods for controlling the shapes and sizes of nanostructures use base-pairing between complementary DNA strands to self-assemble nanostructures from DNA and nanoparticles. DNA Brick-based self-assembly is a particularly useful method for creating DNA nanostructures. It offers a large amount of control over the final shapes and sizes because it uses building blocks that are anisotropic and have predictable geometry. However, this control has not been extended to the self-assembly of nanostructures from nanoparticles. Applying DNA Brick based self-assembly to the self-assembly of …

Gcn5 Impacts Fgf Signaling At Multiple Levels And Activates C-Myc Target Genes During Early Differentiation Of Embryoid Bodies, Li Wang

Dissertations and Theses (Open Access)

Precise control of gene expression during development is orchestrated by transcription factors, signaling pathways and co-regulators, with complex cross-regulatory events often occurring. Growing evidence has identified chromatin modifiers as important regulators for development as well, yet how particular chromatin modifying enzymes affect specific developmental processes remains largely unclear. Embryonic stem cells (ESCs) are self-renewing, pluripotent, and have the abilities to generate almost all cell types in adult tissues. The dual capacity of ESCs to self-renew and differentiate offers unlimited potential for studying gene regulation events at specific developmental stages in vitro that parallel developmental events during embryogenesis in vivo. …

The Dlk1-Meg3 Locus In Malignant Cells Of Proposed Primordial Germ Cell Origins., Zachariah Payne Sellers

Electronic Theses and Dissertations

Primordial germ cells (PGCs) are hypothesized to deposit hematopoietic stem cells (HSCs) along their migration route through the embryo during the early stages of embryogenesis. PGCs also undergo global chromatin remodeling, including the erasure and reestablishment of genomic imprints, during this migration. While PGCs do not spontaneously form teratomas, their malignant development into germ cell tumors (GCTs) in vivo is often accompanied by the retention of hypomethylation at the IGF2-H19 imprinting control differentially methylated region (DMR). Previous studies in bimaternal embryos determined that proper genomic imprinting at two paternally imprinted loci was necessary for their growth and development: Igf2-H19 and …

Preclinical Development Of Therapeutic Strategies Against Triple-Negative And Inflammatory Breast Cancer, Angie M. Torres-Adorno

Dissertations and Theses (Open Access)

Triple-negative (TNBC) and inflammatory (IBC) breast cancer are the most aggressive forms of breast cancer, accounting for 20% and 10% of cancer-related deaths, respectively. Among IBC cases, 30% are additionally classified with TNBC molecular pathology, a diagnosis that significantly worsens patient’s prognosis. The current lack of TNBC and IBC molecular understanding prevents the development of effective therapeutic strategies. To identify effective treatments, we explored aberrant apoptosis pathways and cell membrane fluidity as novel therapeutic targets.

We first identified an effective therapeutic strategy against TNBC and IBC by pro-apoptotic protein NOXA-mediated inhibition of the anti-apoptotic protein MCL1 following inhibition of histone …

The Structural And Functional Properties Of A Double Mutant Of Human Acidic Fibroblast Growth Factor (Hfgf-1), Arwa Mohammed Alghanmi

Graduate Theses and Dissertations

Human acidic Fibroblast Growth Factor 1 (FGF-1), a member of the FGF superfamily, is a potent mitogen and heparin-binding protein involved in a broad spectrum of biological processes, including angiogenesis, cell proliferation, and wound healing. Design of hFGF-1 with an increased thermal stability and an enhanced cell proliferation activity is highly desired for wound healing applications. Herein, we have designed the variant of FGF-1 by substituting two important amino residues in the heparin-binding pocket. The variant was overexpressed in Escherichia coli and was successfully purified to homogeneity using an affinity chromatographic procedure. Far-UV circular dichroism spectroscopic analysis showed that the …

Regulation Of Extracellular Signal-Regulated Kinase By Histone Deacetylase 6, Jheng-Yu Wu

USF Tampa Graduate Theses and Dissertations

Extracellular signal-regulated kinases 1/2 (ERK1/2) are important kinases regulating cell proliferation and cell migration, and have been established as therapeutic targets for cancer treatment. Previously, we found that ERK1 phosphorylates histone deacetylase 6 (HDAC6) to regulate its enzymatic activity. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we have discovered that ERK1/2 are acetylated proteins and shown that HDAC6 manipulates ERK1’s kinase activity via deacetylation. We demonstrated that both ERK1 and ERK2 interact with HDAC6 physically. We showed that the acetylation level of GST-ERK1/2 increased in a dose- and time-dependent manner upon treatment with a pan-HDAC inhibitor, Trichostatin …

The General Amino Acid Permease Gap1 Is Regulated Differentially By Torc1 Activation And Inhibition, Ray Bowman

D.U.Quark

How does cell signaling in response to extracellular stressors impact the trafficking of membrane proteins? In particular, the TORC1 complex plays a key role in this process and while some details of this system have reported, in a recent Journal of Biological Chemistry publication, Andre’s group has revealed new details of this pathway focusing on the general amino acid permease Gap1 as a model cargo. Andre et al. describe a novel and distinct pathway wherein ubiquitylation and downregulation of Gap1 is regulated not only by amino acid-induced activation of TORC1, but also by numerous sources of TORC1 inhibition and cellular …

Spatiotemporal Regulation Of Atg1 Kinase Activation In Selective Autophagy, Ning Sun

D.U.Quark

Autophagy is a potent intracellular degradation system and thus its activation requires exquisite regulation to maintain cellular homeostasis. Atg1, a serine-threonine protein kinase, is essential in both selective and non-selective autophagy. New findings suggest that in selective autophagy, Atg1 is activated at the vacuole by convergence of two independent recruitment pathways to prevent aberrant autophagy induction.

Hsp70 Conformational Plasticity Allows For Expansive Chaperone Role, Megan Bean

D.U.Quark

The Hsp70 system is an essential component of chaperone activity in many organisms. Hsp70 functions include: protein folding, aggregation prevention, trafficking, and enzyme regulation. Hsp70’s ability to bind such a vast array of substrates suggests wide range of conformational plasticity. By utilizing a single mode optical tweezers technique, Mashaghi1 et al., confirms previous theories Hsp70 binds and stabilizes extended peptide segments but also partially folded and near-native protein.

Plasmodium Falciparum 26s Proteasome Network: A Mystery Solved, Christina Grogan

D.U.Quark

One ofthe most devastating diseases thatthreatens the world population is malaria. The 26S proteasome complex of the malaria parasite Plasmodium falciparum, which was previously unknown, was characterized by the authors through an affinity purification protocol that isolated functional 26S proteasome complexes. This allowed for the identification of subunit composition and PfUSP14, a proteasomeassociated deubiquitinase. This new understanding presents a potential target to disrupt protein regulation in thequest for effective antimalarial strategies.

Autophagy Inhibition In Pain: Role Of A Microrna, Andrea Stevens

D.U.Quark

Neuropathic pain caused by peripheral nerve injury (PNI) leads to the activation and infiltration of microglial cells and to a neuroinflammatory-induced pain state. miRNAs and autophagy are two main factors and/or mechanisms which have the ability to alter the pain state. In this study, miR-195 was shown to be markedly increased after PNI and associated with the pain phenotype. In addition, inhibition of autophagy in vivo led to p62 accumulation, decreased production of LC3, and inhibition of ATG14.

A Lipid Binding Structure And Functional Analysis Of Human Arv1, Jessie Lee Cunningham

Graduate School of Biomedical Sciences Theses and Dissertations

Metabolic Syndrome (MetS) is a combination of risk factors that can over time increase the probability of developing diseases, including cardiovascular disease, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). Acyl-coenzyme-A: cholesterol O-acyl transferase related enzyme required for viability-1, abbreviated as Arv1, is an evolutionarily conserved putative lipid binding protein. Several studies have implicated hArv1 as a critical regulator of lipid transport and trafficking.

Recent work using an Arv1 knock out (KO) mouse model have established a clear link between Arv1 function and the progression of MetS and NAFLD/NASH [unpublished data] [1]. Overall, studies show that …

Functional Roles Of Matrix Metalloproteinases In Bone Metastatic Prostate Cancer, Jeremy S. Frieling

USF Tampa Graduate Theses and Dissertations

Skeletal metastasis is a lethal component of many advanced cancers including prostate, the second most common cancer among men. Patients whose prostate cancer is localized and detected early benefit from multiple treatment options ranging from active surveillance to radiation and surgery, resulting in a 5-year survival rate of nearly 100%. Unfortunately, the prognosis and survival for patients with advanced metastatic disease is much worse due to the highly aggressive nature of the disease and a paucity of treatment options. Understanding the mechanisms and interactions that occur between metastatic cancer cells and the bone will enable the future treatment landscape for …

Applied Drug Development And Combinatorial Strategies For Antimicrobial Treatment, Steven K. Lai Hing

Andrews Research Conference

Streptococcus mutans JH1140 is a strain of bacteria which produces a lantibiotic product, named mutacin 1140. Mutacin 1140 has been shown to be effective at inhibiting Gram-positive bacterial infections caused by Staphylococcus aureus and Streptococcus pneumoniae. Mutacin 1140 is a ribosomally synthesized peptide antibiotic that undergoes extensive posttranslational modifications (PTM). We have found that Mutacin 1140 and an aminoglycoside, Kanamycin, when combined together, act synergistically against Staphylococcus aureus. This was determined by performing serial kill curve dilution overlays on solid media, followed up with kill curve by microdilution plate, and most recently confirmed with kill curve CFU count plates …