Molecular Biology Commons^™

The Role Of Secreted Proteases In Regulating Disease Progression In Staphylococcus Aureus, Brittney D. Gimza

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus is a highly successful pathogen capable of producing a wealth of virulence factors in the human host. Of note, ten extracellular proteases are produced alongside these virulence factors and play a multifaceted role during infection. They not only cleave host proteins to promote bacterial invasion, immune evasion and survival, but also control disease progression by modulating the stability of self-derived pathogenic determinants. The importance of the secreted proteases modulating virulence factor stability is evidenced by our groups previous finding that a protease-null strain has a substantially increased infectious capacity in a murine model of sepsis; resulting from the …

A Novel Cytokine Response Modulatory Function Of Mek Inhibitors Mediates Therapeutic Efficacy, Mengyu Xie

USF Tampa Graduate Theses and Dissertations

Despite the recent success of immune-checkpoint blockade therapy for late-stage non-small cell lung cancer (NSCLC), lung cancer is still the leading cause of cancer deaths worldwide. One of the most important characteristics of lung cancer in therapeutic decision-making are the targetable molecules, including EGFR, ALK, BRAF, and MEK. The excitement of immune-checkpoint blockade therapy has triggered concerted efforts that focus on exploring combinations of immune checkpoint therapy with other approved therapeutic regimens aiming at further augmenting positive outcomes and survival. However, the lack of understanding of underlying mechanisms and evidence-based clinical testing has hindered the progress to a cure. Hence, …

Modeling Of Interaction Of Ions With Ether- And Ester-Linked Phospholipids, Matthew W. Saunders

USF Tampa Graduate Theses and Dissertations

Phospholipids are present in all parts of cells and are used in many signalling and struc- tural roles. As structural molecules they act as the main component of cellular membranes. Bilayer properties are heavily influenced by the structure of their component polar lipids, and different lipids are found in different organisms. A distinguishing feature of Archaeal plasma membranes is that their phospholipids contain ether-links, as opposed to bacterial and eukaryotic plasma membranes where phospholipids primarily contain ester-links. In our work we examine the effects of salt on bilayer structure in the case of both ester- and ether-linked lipid bilayers. We …

Design And Delivery Of Synthetic Mrna By A Peptide Nanoparticle, John H. Lockhart

USF Tampa Graduate Theses and Dissertations

The field of synthetic mRNA therapeutics is a rapidly expanding arm of gene therapies. The use of mRNA provides multiple benefits over viral or DNA vectors. synthetic mRNA vectors are immediately translated into protein after entering the cytoplasm of cells in contrast to DNA vectors that must first be transcribed to mRNA in the nucleus. This allows synthetic mRNA to produce a therapeutic protein in any cell type, including non-dividing cells. In addition, the non-replicative nature of mRNA means that insertional mutagenesis or generation of escape mutants is not a concern. However, the stimulation of innate immune responses by unmodified …

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

USF Tampa Graduate Theses and Dissertations

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

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

USF Tampa Graduate Theses and Dissertations

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

Targeting Pten For Therapy In Cancer And Ptenopathies, Emily Palumbo

USF Tampa Graduate Theses and Dissertations

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

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

USF Tampa Graduate Theses and Dissertations

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

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

USF Tampa Graduate Theses and Dissertations

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

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

Novel Insights Into The Multifaceted Roles Of Blm In The Maintenance Of Genome Stability, Vivek M. Shastri

USF Tampa Graduate Theses and Dissertations

Genomic instability is a hallmark of disorders in which DNA replication and repair genes are dysfunctional. The tumor suppressor RECQ helicase gene BLM encodes the 3’-5’ DNA Bloom syndrome helicase BLM, which plays important roles during DNA replication, recombination and repair to maintain genome stability. Mutations within BLM cause Bloom syndrome, an autosomal recessive disorder characterized by growth defects, immunodeficiency, >10-fold higher sister chromatid exchange compared to normal cells, and an increased predisposition to a wide range of cancers from an early age. Single nucleotide polymorphisms or SNPs in BLM have been reported to be associated with susceptibility to a …

Advanced Proteomic And Epigenetic Characterization Of Ethanol-Induced Microglial Activation, Jennifer Guergues Guergues

USF Tampa Graduate Theses and Dissertations

Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes and have been implicated in several diseases and disorders of the central nervous system. Here, we described a method optimized for sensitive and rapid quantitative proteomic analysis of microglia that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a microglial cell count expected from an individual mouse brain (~300K) while also simultaneously providing the first comprehensive proteomic characterization of a novel adult-derived mouse microglial cell line. This enhanced method was used throughout all subsequent works and was especially necessary when we …

Investigating The Role Of The Chromosome 19 Microrna Cluster In Human Trophoblast Differentiation And Infantile Hemangioma, Ezinne Francess Mong

USF Tampa Graduate Theses and Dissertations

Trophoblast differentiation and invasion is essential for normal implantation and establishment of the maternal-fetal interface, which allows for proper nutrient exchange and support of the fetus. For this to occur, cytotrophoblasts must undergo an epithelial to mesenchymal transition and differentiate into migratory and invasive extravillous trophoblasts (EVTs) that invade the maternal decidua and myometrium. Trophoblast differentiation, migration and invasion is highly regulated by a complex network of signaling pathways, adhesion molecules and transcription factors and is important for the remodeling of maternal spiral arteries from low flow, high resistance to high flow, low resistance vessels to allow optimal perfusion of …

Role Of Oncogenic Protein Kinase C-Iota In Melanoma Progression; A Study Based On Atypical Protein Kinase-C Inhibitors, Wishrawana Sarathi Bandara Ratnayake

USF Tampa Graduate Theses and Dissertations

Irrespective of plentiful efforts to enhance primary prevention and early detection, the number of melanoma cases in the United States has increased steadily over the past 30 years, thus greatly affecting public health and the economy. We have investigated the effects of five novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD), 3,4-Diaminonaphthalene-2,7-disulfonic acid (DNDA), [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocyte cell lines. Molecular docking data suggested that both ACPD and DNDA specifically bind to protein kinase …

Conserved Glycine Residues Control Transient Helicity And Disorder In The Cold Regulated Protein, Cor15a, Oluwakemi Sowemimo

USF Tampa Graduate Theses and Dissertations

COR15A is a cold regulated disordered protein from Arabidopsis thaliana that contributes to freezing tolerance in plants by protecting membranes. It belongs to the (LEA) Late Embryogenesis Abundant group of proteins that accumulate during the later stage of seed development and are expressed in various parts of the plant. During freezing-induced cellular dehydration, COR15A transitions from a disordered structure to a mostly α-helical structure that binds and stabilizes chloroplast membranes when cells dehydrate due to freezing. We hypothesize that increasing the transient α-helicity of COR15A under normal conditions will increase its ability to bind and protect chloroplast membranes when cells …

Biochemical And Proteomic Approaches To Determine The Impact Level Of Each Step Of The Supply Chain On Tomato Fruit Quality, Robert T. Madden

USF Tampa Graduate Theses and Dissertations

Fresh fruits and vegetables (FFVs) are the most frequently wasted foods because of their perishability and handling requirements. However, there is a lack of information on how much each step of the supply chain impacts FFVs quality, particularly on tomatoes, and what measures need to be taken for an immediate and effective impact on waste reduction. There is also no information on how the supply chain affects the proteome of the tomato and what proteins are differentially regulated by the most impactful steps of the supply chain. The objectives of the work presented on this thesis were to evaluate the …

Integrated Molecular Characterization Of Lung Adenocarcinoma With Implications For Immunotherapy, Nicholas T. Gimbrone

USF Tampa Graduate Theses and Dissertations

This dissertation covers a variety of the genetic and molecular abnormalities of lung adenocarcinoma with an emphasis on STK11 loss and its implications on immunotherapy response. Given that lung cancer is the leading cancer killer, novel therapies are in great demand. In particular, immunotherapy has shown some of the most promise in the last decade but remains limited due to nearly 80% of patients not significantly responding. This dissertation aims to molecularly characterize lung adenocarcinoma while attempting to explain the reason why patients with STK11 loss do not respond to immunotherapy.

In the first chapter we discuss the relationship between …

Cyclophilin 40 As A Novel Disaggregase, Jeremy Dustin Baker

USF Tampa Graduate Theses and Dissertations

The negative health and economic impacts of neurodegenerative diseases on Americans is astounding and accelerating with an aging population. The Alzheimer’s Association reports that 5.7 million Americans suffer from Alzheimer’s disease (AD), a number which is expected to increase to 14 million by 2050. In economic terms, AD and other neurodegenerative disorders will cost the US over $275 billion in 2018, rising to over $1 trillion annually by 2050. AD causes gross brain atrophy and is most damaging throughout the cortex and the hippocampus, regions required for higher cognitive function and memory. AD presents as tangles within neurons composed of …

Actinomycin D And Telmisartan Combination Therapy Targets Lung Cancer Stem Cells, Ryan Green

USF Tampa Graduate Theses and Dissertations

The failure of lung cancer treatments has been attributed partly to the development of drug resistance, however the underlying cellular and molecular mechanisms are poorly understood. It has been suggested that a very small group of specific cells within the heterogeneous tumors, cancer initiating stem cells (CSC), develop resistance to treatment, survive and later initiate the growth of new tumors. Due to their pivotal role in maintenance and relapse of tumors following the acquisition of drug resistance, we reasoned that novel drugs targeting cancer cells and CSC might provide the most effective treatments, if not a cure. To this end, …

Investigating The Roles Of Fucosylation And Calcium Signaling In Melanoma Invasion, Tyler S. Keeley

USF Tampa Graduate Theses and Dissertations

Melanoma is the deadliest form of skin cancer. Prognosis for early stage melanoma patients is excellent, and surgery is often curative for these patients. However, once patients have presented with invasive disease, the average 5-year survival rate drops significantly from over 90% to between 10 and 15%. Several therapies have been developed to target a commonly mutated oncogene BRAF, or its downstream effectors. Unfortunately, while these treatments show robust initial response, most patients relapse within a year. Moreover, therapy-resistant tumors are often more invasive and metastatic. Therefore, it is important to investigate the molecular mechanisms underlying melanoma invasion and metastasis, …

Investigating The Role Of The Rna Binding Protein Lin28 In The Human Placenta: Implications For Preeclampsia, John Canfield

USF Tampa Graduate Theses and Dissertations

An essential event during early pregnancy is the invasion of trophoblasts into the maternal decidua, which is necessary for proper implantation and establishment of maternal-fetal interface and ultimately allows for proper nutrient exchange and immunological tolerance of the growing fetus. For this invasion to occur, cells originating from the trophectoderm undergo an epithelial to mesenchymal transition to become invasive extravillous trophoblasts and begin invading the uterine decidual tissue. Through the secretion of matrix metalloproteinases and through interactions with many cytokines and cell-adhesion molecules, this well-orchestrated process of trophoblast invasion results in extensive remodeling of the maternal spiral vasculature by the …

Myc Distant Enhancers Underlie Ovarian Cancer Susceptibility At The 8q24.21 Locus, Anxhela Gjyshi Gustafson

USF Tampa Graduate Theses and Dissertations

Ovarian cancer is a leading cause of death among women diagnosed with cancer. Mortality rate is high because an overwhelming majority of new cases are diagnosed with late-stage disease when the survival statistics are very poor with an overall 5-year survival rate of less than 40%. Despite the large burden of disease, the etiology of ovarian cancer is not well understood. In addition to linkage studies that have identified highly penetrant cancer susceptibility genes such as BRCA1 and BRCA2, the emergence of Genome Wide Association Studies (GWAS) in the last decade has facilitated the identification of common genetic variants with …

A Systems Chemical Biology Approach For Dissecting Differential Molecular Mechanisms Of Action Of Clinical Kinase Inhibitors In Lung Cancer, Natalia Junqueira Sumi

USF Tampa Graduate Theses and Dissertations

Lung cancer is the second most common cancer type and is associated with high mortality rates. The survival rate for lung cancer patients has increased slowly in the last decade mainly as the result of the development of novel targeted and immune therapies. However, non-small cell lung cancer patients lacking known or actionable driver mutations and small cell lung cancer patients with recurrent disease are still in urgent need of new therapies. Drug repurposing is an efficient way to identify new therapies since it uses clinically relevant small molecule drugs. Determination of off-targets of small molecules is a novel approach …

Investigation Of Alcohol-Induced Changes In Hepatic Histone Modifications Using Mass Spectrometry Based Proteomics, Crystina Leah Kriss

USF Tampa Graduate Theses and Dissertations

Alcohol liver disease (ALD) is a major health concern throughout the world. Currently, in the United States, 17 million people suffer from alcoholism, of which 1.4 million people are receiving treatment [1, 2]. The link between ethanol metabolism, reactive oxygen species (ROS) and liver injury in ALD has been well characterized over the last couple decades [3-10]. Ethanol metabolism relies on the availability of the cofactor NAD+ for the oxidation of ethanol into acetate, consequently causing alterations in redox potential. Redox dysfunction within the mitochondria can affect multiple pathways important in maintaining cellular homeostasis. Chapter 1 provides an introduction to …

The Role Of Phosphohistidine Phosphatase 1 In Ethanol-Induced Liver Injury, Daniel Richard Martin

USF Tampa Graduate Theses and Dissertations

Chronic liver diseases, which includes alcoholic liver disease (ALD), are consistently among the top 15 leading causes of death in the United States. ALD is characterized by progression from a normal liver to fatty liver disease (hepatic steatosis), which can lead to cirrhosis, alcoholic hepatitis, and liver failure. We have identified a novel role of phosphohistidine signaling, mediated through phosphohistidine phosphatase 1 (PHPT1), in the onset of hepatic steatosis. We have identified PHPT1 as a target of selective oxidation following acute ethanol exposure as well as being downregulated following chronic ethanol exposure. We mapped the oxidative modification site and developed …

Elastin-Like Polypeptide Fusion Tag As A Protein-Dependent Solubility Enhancer Of Cysteine-Knot Growth Factors, Tamina L. Johnson

USF Tampa Graduate Theses and Dissertations

Elastin-like peptide (ELP) fusions promote therapeutic delivery and efficacy. Recombinant proteins, like neurotrophins, lack bioavailability, have short in vivo half-lives, and require high manufacturing costs. Fusing recombinant proteins with genetically encodable ELPs will increase bioavailability, enhance in vivo solubilization, as well as provide a cost-effective method for purification without the need for chromatography. During expression of neurotrophin-ELP (N-ELP) fusions, dense water-insoluble aggregates known as inclusion bodies (IBs) are formed. Inclusion bodies are partially and misfolded proteins that usually require denaturants like Urea for solubilization. Strong denaturants arrest ELPs stimuli-responsive property and increase unwanted aggregation, making purification difficult, yet possible. The …

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 …

Mechanism Elucidation And Inhibitor Discovery Against Serine And Metallo-Beta-Lactamases Involved In Bacterial Antibiotic Resistance, Orville A. Pemberton

USF Tampa Graduate Theses and Dissertations

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes …

Cell Division Regulation In Staphylococcus Aureus, Catherine M. Spanoudis

USF Tampa Graduate Theses and Dissertations

Cell division is a fundamental biological process that occurs in all kingdoms of life. Our understanding of cell division in bacteria stems from studies in the rod-shaped model organisms: Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The molecular underpinnings of cell division regulation in non-rod-shaped bacteria remain to be studied in detail. Rod-shaped bacteria possess many positive and negative regulatory proteins that are essential to the proper placement of the division septa and ultimately the production of two identical daughter cells, many of which are absent in cocci. Given that essential cell division proteins are attractive antibacterial drug targets, it …

Microfluidic Biopsy Trapping Device For The Real-Time Monitoring Of The Tumor Microenvironment, Angela Holton

USF Tampa Graduate Theses and Dissertations

The tumor microenvironment is composed of cellular and stromal components such as tumor cells, mesenchymal cells, immune cells, cancer associated fibroblasts and the supporting extracellular matrix. The tumor microenvironment provides crucial support for growth and progression of tumor cells and affects tumor response to therapeutic interventions. To better understand tumor biology and to develop effective cancer therapeutic agents it is important to develop preclinical platforms that can faithfully recapitulate the tumor microenvironment and the complex interaction between the tumor and its surrounding stromal elements. Drug studies performed in vitro with conventional two-dimensional cancer cell line models do not optimally represent …