Molecular Biology Commons^™

Targeting Bet Proteins Downregulates Mir-33a To Promote Synergy With Pim Inhibitors In Cmml, Christopher T. Letson

USF Tampa Graduate Theses and Dissertations

Chronic Myelomonocytic Leukemia (CMML) is a rare myeloid malignancy with a dismal prognosis and no therapeutic options which are capable of altering the natural course of the disease. There remains a significant need for novel therapies that are able to meaningfully improve patient outcomes. In this study we explore the effectiveness of Bromodomain and Extra-Terminal domain protein inhibitor (BETi) combinations in CMML. Preclinical studies in myeloid neoplasms have demonstrated efficacy of BETi. However, BETi demonstrate poor single agent activity in clinical trials. Several studies suggest that combinations with other anti-cancer inhibitors may enhance the efficacy of BETi. To nominate BETi …

Thermodynamic Frustration Of Tad2 And Prr Contribute To Autoinhibition Of P53, Emily Gregory

USF Tampa Graduate Theses and Dissertations

The intrinsically disordered transcription factor and tumor suppressor p53 binds to promoter response element DNA upon cellular stress and activates genes associated with cell cycle arrest, senescence, and apoptosis. Disruption of sequence specific binding to target gene promoters is heavily implicated in human health, where a majority of cancers contain mutations localized to the DNA binding domain (DBD) of p53. p53 DNA binding is regulated by posttranslational modifications, associations with cellular factors, and by an autoinhibitory intramolecular interaction. The autoinhibitory intramolecular interaction occurs when the disordered N-terminal transactivation domain (TAD) interacts with the ordered DBD. Previous work in the Daughdrill …

Withaferin A And Immune Checkpoint Blocker Therapy For The Treatment Of Non-Small Cell Lung Cancer, Roukiah Khalil

USF Tampa Graduate Theses and Dissertations

Lung cancer is the first cause of cancer-related deaths in both men and women with an overall five-year survival rate of 28%. Although immune checkpoint blockers (ICBs) are currently FDA-approved for the treatment of non-small cell lung cancer (NSCLC), only 17-20% of patients achieve durable responses by the induction of immunologic memory. The lack of response in most patients can be attributed to the tumor-intrinsic or tumor-extrinsic immune resistance mechanisms. A biomarker of importance is the Programmed Death Ligand-1 (PD-L1), as higher PD-L1 expression is usually associated with a better response to ICBs. Although studies have attempted to combine ICBs …

Exploring Strain Variation And Bacteriophage Predation In The Gut Microbiome Of Ciona Robusta, Celine Grace F. Atkinson

USF Tampa Graduate Theses and Dissertations

Current microbiome studies have shown that the maintenance of homeostasis betweenmicrobial populations (e.g. bacteria, viruses) and the host immune system (e.g. innate immune molecules) is necessary for balancing health and disease outcomes within the host. These studies most often utilize vertebrate models; however, research in this field can benefit from diverse model systems that facilitate our ability to conduct experiments to identify phylogenically conserved rules influencing homeostasis in the gut of animals. The Dishaw has developed the use of a filter-feeding marine invertebrate chordate, Ciona robusta, to model such fundamental interactions[1]–[6]. While most biological diversity and functional contribution within microbiomes …

A Novel Role For Enos In Regulating Lymphatic Valve Development During Embryogenesis, Drishya Iyer

USF Tampa Graduate Theses and Dissertations

Lymphedema is a disease that occurs when lymph flow is impaired, resulting in tissue swelling, fibrosis, chronic inflammation and recurrent secondary infections. Lymphatic valves play a critical role in maintaining unidirectional lymph flow and evidence for valve defects have been reported in lymphedema patients. The lack of drugs that can correct lymphatic valve defects warrants a better understanding of the molecular regulators of lymphatic valve development and maintenance. Lymphatic valves first develop during embryogenesis in response to mechanotransduction signaling pathways triggered by oscillatory lymph flow. Since eNOS (gene name: Nos3) is a well characterized mechanotransduction signaling molecule in blood vessels, …

X-Linked Ubiquitin-Specific Peptidase 11 Increases Tauopathy Vulnerability In Women, Yan Yan

USF Tampa Graduate Theses and Dissertations

Women experience significantly higher tau burden and increased risk for Alzheimer’s disease (AD) than men, yet the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially …

Regulation Of The Heat Shock Response Via Lysine Acetyltransferase Cbp-1 And In Neurodegenerative Disease In Caenorhabditis Elegans, Lindsey N. Barrett

USF Tampa Graduate Theses and Dissertations

The decline of proteostasis is a hallmark of aging that is, in part, affected by the dysregulation of the heat shock response (HSR), a highly conserved cellular response to proteotoxic stress in the cell. The heat shock transcription factor HSF-1 is well-studied as a key regulator of proteostasis, but mechanisms that could be used to modulate HSF-1 function to enhance proteostasis during aging are largely unknown. In this study, we examined lysine acetyltransferase regulation of the HSR and HSF-1 in C. elegans. We performed an RNA interference screen of lysine acetyltransferases and examined mRNA expression of the heat-shock inducible gene …

A Protein-Based Therapeutic Combination For The Treatment Of Hard-To-Heal Wounds, Graham L. Strauss

USF Tampa Graduate Theses and Dissertations

Chronic wounds present many clinical challenges in relation to the successful treatment and closure of the damaged tissue. Most current treatment methods focused on one or two aspects to drive wound closure, while most chronic wounds are multifactorial environments with many of those dependencies relying on the termination of one another to effectively gain tissue construction, closure, and full skin thickness and composition. Natural wound healing processes allude to potential biologics that can impede the chronic breakdown of tissue, while restoring deposition of new tissue, and effectively leading to a healed wound. Proteases secreted by the body’s immune system lay …

The Role Of Eicosanoid Metabolism In Mammalian Wound Healing And Inflammation, Kenneth D. Maus

USF Tampa Graduate Theses and Dissertations

Inflammatory wounds, both chronic and acute, lead to increased morbidity and mortality rates, especially in the elderly population. The annual healthcare cost for chronic wound care alone is over $39B in the US and the demographic of susceptible patients is steadily increasing due to an aging population and lifestyle-related diseases (e.g., hyperlipidemia, obesity, and type 2 diabetes). In fact, many chronic wounds currently have a worse 5-year outlook than certain types of cancers. This shows the need for expediting the wound healing process in such a way that compresses inflammatory signaling and encourages wound resolution without sacrificing pathogen removal and …

Role Of Bmi1 In Acute Lung Injury, María Helena Hernández-Cuervo

USF Tampa Graduate Theses and Dissertations

Acute Lung Injury (ALI) is a set of signs and symptoms that lead to acute hypoxemic respiratory failure characterized by bilateral pulmonary infiltrates not attributed to cardiogenic origin. It is caused by a massive innate immune response, with the migration of white blood cells (neutrophils and macrophages principally) and a cytokine storm, followed by alterations in mitochondrial function, increase in reactive oxygen species production, and oxidative stress that in turn induces more mitochondrial damage. Several studies have shown that mitochondrial alterations are key events in the mechanism of ALI and reducing mitochondrial dysfunction could be a possible target in the …

Unraveling The Role Of Novel G5 Peptidase Family Proteins In Virulence And Cell Envelope Biogenesis Of Staphylococcus Aureus, Stephanie M. Marroquin

USF Tampa Graduate Theses and Dissertations

Virulence factors and the bacterial cell envelope are two important components in S. aureus pathogenesis and survival. More importantly, understanding the regulation of these cellular processes is crucial to further understanding and combating this successful pathogen. To date, numerous factors have been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane G5 peptidase family protein, as a novel effector of this system. Using a USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis, and …

The Role Of Cpeb2 Alternative Splicing In Tnbc Metastasis, Shaun C. Stevens

USF Tampa Graduate Theses and Dissertations

Breast cancer is the second leading cause of cancer-related deaths for women in the U.S. Although the overall 5-year survival rate for breast cancer is 90%, this rate drops substantially for triple-negative breast cancer (TNBC) due to its high metastatic potential. Furthermore, there is a lack of targeted therapeutics for TNBC, and clinical trials have been largely unsuccessful. These characteristics validate the need for identifying novel therapeutic targets for the treatment of TNBC. The study of alternative splicing (AS) has emerged as a powerful tool to elucidate the molecular underpinnings driving cancer.

Our lab has identified cytoplasmic polyadenylation element-binding protein …

Investigation Of An Alternative Protocol For The Production Of Sars-Cov-2 Antigenic Proteins, Nichole Ninaltowski

USF Tampa Graduate Theses and Dissertations

With the COVID-19 pandemic showing no signs of slowing down, large-scale antigenic protein production is still needed for surveillance using serologic assays. From screening to vaccines to biotherapeutics, being able to produce the proteins for these assays is essential; however, the current gold standard method for producing SARS-CoV-2 spike proteins is prohibitively expensive for most research groups.

Alternative methods of transfecting mammalian cells to produce recombinant proteins that are relatively inexpensive have been used for years. Unlike the expensive, commercially available lipid-based methods, other established methods such as polyethyleneimine (PEI), are considerably easier, and cheaper to meet the needs of …

Cellular And Molecular Alterations Associated With Ovarian And Renal Cancer Pathophysiology, Ravneet Kaur Chhabra

USF Tampa Graduate Theses and Dissertations

Elucidating molecular alterations underlying tumor development and chemoresistance are critical to expand our understanding of the disease pathophysiology. This dissertation is focused on analyzing the cellular and molecular alterations associated with LPA-induced chemoresistance in clear cell renal cell carcinoma (ccRCC) cells and chronic iron-induced deregulation of miRNA expression in fallopian tube secretory epithelial cells (FTSECs).

Kidney cancer is one of the ten most common cancers worldwide with <15% survival rate at advanced stage (American Cancer Society). ccRCC is the most common type of kidney cancer and is described as a metabolic disease characterized by deregulated lipid metabolism leading to increased intracellular lipid droplets [9, 10]. The current molecular-targeted treatment strategies involve VEGF/VEGFR and mTOR inhibition [9, 12]. However, there are limitations to these approaches leading to the reduced efficacy and/or increased resistance in ccRCC cells [13, 14]. Therefore, it is important to decipher the factors involved in compromising the chemosensitivity in these cells.

Lysophosphatidic acid (LPA), a bioactive phospholipid, was previously reported to increase resistance against Sunitinib (VEGFR/PDGFR inhibitor) in ccRCC cells and to increase migration and invasion in various tumors [15-17]. In Chapter 3 of …

Screening Next-Generation Fluorine-19 Probe And Preparation Of Yeast-Derived G Proteins For Gpcr Conformation And Dynamics Study, Wenjie Zhao

USF Tampa Graduate Theses and Dissertations

GPCR regulates numerous diverse physiological processes relevant to diabetes, obesity, Alzheimer's diseases, and several central nervous system disorders and targets proteins in signaling pathways. It has created nearly 200 billion profits from its derivative drugs in 2018. There are near 400 structures of over 70 GPCRs have been resolved by X-ray crystallography, cryo-electron microscopy, and NMR spectroscopy. One of the current challenges that remain in the conformational transition and dynamics study using NMR spectroscopy is to obtain sufficient quantities of the G proteins and GPCRs. Pichia pastoris has shown its tremendous promise in expressing the GPCRs in a high yield, …

Evolution Of Targeted Therapy Resistance In Eml4-Alk Positive Non-Small Cell Lung Cancer, Robert Vander Velde

USF Tampa Graduate Theses and Dissertations

Targeted therapies have emerged as potent treatments that lead to the remission of many tumors. However, they rarely cure cancers in advanced, metastatic settings. This is due to the evolution of resistance, which in turn can be ascribed to the survival of small subpopulations of tolerant and/or resistant cells. Here we investigated the evolution of resistance to EML4-ALK inhibitors in non-small cell lung cancer (NSCLC) and demonstrated that resistance evolves gradually, from unique pre-treatment sub-populations, as multiple resistance mechanisms accumulate in a Darwinian fashion. Despite accumulating multiple changes, cells evolved, in parallel, toward similar inhibitor specific phenotypes. Evolving cells have …

Transcriptomic And Functional Investigation Of Bacterial Biofilm Formation, Brooke R. Nemec

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus and Acinetobacter baumannii are two highly successful human pathogens, which have adopted very different, but effective survival strategies. The success of S. aureus is attributed to the tight regulation of an arsenal of virulence factors. Conversely, A. baumannii lacks what would be considered traditional virulence factors and, instead, has developed a high tolerance for environmental stress, which allows it to persist in unforgiving environments, including nosocomial settings and the human body. One common characteristic of these two organisms is their proclivity for biofilm formation. Herein, we discuss the diverse mechanisms governing biofilm formation for A. baumannii and S. …

Cytoplasmic Polyadenylation Element Binding Protein 2 Alternative Splicing Regulates Hif1Α During Chronic Hypoxia, Emily M. Mayo

USF Tampa Graduate Theses and Dissertations

Chronic pulmonary hypoxia commonly results in the sustained expression of HIF1 (hypoxia inducible factor 1), a heterodimeric transcription factor, that, if unrestrained, can result in dramatic vasculature remodeling, pulmonary arterial hypertension, and right-sided heart failure. Together, these pulmonary disorders cost approximately $100 billion annually to treat due to the limited therapeutic targets designed to inhibit HIF1 expression. In this study, we introduce a translational regulator of HIF1 expression, known as Cytosolic polyadenylation element binding proteins 2 (CPEB2). Our lab has previously demonstrated in cancer cells that alternatively spliced isoforms of CPEB2 regulate the translation of the HIF1 oxygen-dependent subunit, HIF1α, …

Circrev1 Expression In Triple-Negative Breast Cancer, Meagan P. Horton

USF Tampa Graduate Theses and Dissertations

Triple-negative breast cancer (TNBC) comprises only 24% of breast cancer cases, yet is the second leading cause of cancer mortality in women due to its aggressive nature (1). This increase in mortality is due to the lack of receptors for three targetable growth factors (HER2, progesterone, and estrogen receptors). Our previous studies have indicated that these cancers are highly dysregulated in respect to alternative splicing. Hence, we undertook a study aimed at identifying circular RNAs (circRNAs) generated from back-splicing events which were dysregulated in TNBC. We have identified a novel circRNA transcript, circular REV1 (circREV1), which is upregulated in our …

Bone Microenvironmental Control Of Skeletal Malignancy, Chen Hao Lo

USF Tampa Graduate Theses and Dissertations

Bone is a common site of metastasis for many solid malignancies. Bone-metastatic cancers pose a significant clinical problem worldwide and is among the main causes for cancer patient morbidity and mortality. Patients with advanced bone-metastatic diseases often present with either osteolytic or osteogenic bone diseases as their cancers progress. These bone pathologies are products of the cancer co-opting the local bone remodeling stroma to yield important growth nutrients and factors. Unfortunately, skeletal metastases remain incurable and are fatal. Identifying and understanding the causal multicellular and molecular interactions underlying skeletal malignancies can yield crucial ideas for targeting and inhibiting disease progression. …

Posttranslational Modification And Protein Disorder Regulate Protein-Protein Interactions And Dna Binding Specificity Of P53, Robin Levy

USF Tampa Graduate Theses and Dissertations

p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome …

New Mechanisms That Control Fact Histone Chaperone And Transcription-Mediated Genome Stability, Angelo Vincenzo De Vivo Diaz

USF Tampa Graduate Theses and Dissertations

The Role of deubiquitinating enzymes (DUBs) in transcription, replication and genome integrity is not one that has been extensively researched. OTU DUBs are a particular class of enzyme with very little known about them.OTUD5 is a cysteine protease in the OTU family responsible to processing lysine 48 and lysine 63 ubiquitin chains. Recently, it has been implicated in to play a role in transcription through its binding partner UBR5. OTUD5 has also been shown to interact with proteins such as PDCD5 and p53, potentially have great importance in cell fate. In this study, I describe new discovered functions for OTUD5 …

New Mechanisms That Regulate Dna Double-Strand Break-Induced Gene Silencing And Genome Integrity, Dante Francis Deascanis

USF Tampa Graduate Theses and Dissertations

Proliferating cells are constantly threatened by genotoxic stressors that can potentially lead to genomic instability. Breaks in the DNA, namely double-strand breaks, are detrimental sources of damage that must be repaired to maintain genomic integrity and prevent potential tumorigenesis. Here we discuss a gene silencing mechanism flanking damaged chromatin. Gene silencing and transcriptional repression at damaged DNA are necessary to prevent potential genomic aberrations from occurring through conflicts with the DNA repair machinery. BMI1, a core polycomb protein in the polycomb repressive complex 1 (PRC1) has been known to play a role in gene silencing at damaged chromatin. However, the …

Role Of Ceramide-1 Phosphate In Regulation Of Sphingolipid And Eicosanoid Metabolism In Lung Epithelial Cells, Brittany A. Dudley

USF Tampa Graduate Theses and Dissertations

Ceramide 1-Phosphate (C1P) is a sphingolipid metabolite which plays a large role in inflammation, cell survival and proliferation¹. C1P is known to have both pro- and anti-apoptotic roles in lung cancer cells, governed by ceramide kinase (CERK), upstream of precursor ceramide (Cer)². Previous work reveals C1P serves as the liaison between sphingolipid and eicosanoid synthesis, by decreasing the dissociation rate of group IVA cytosolic PLA₂ (cPLA_2α) from the Golgi membrane, C1P directly activates this phospholipase for downstream eicosanoid synthesis and subsequent inflammatory response³. CERK has been discovered to modulate eicosanoid synthesis, …

Novel Long Non-Coding Rna Cdlinc Promotes Nsclc Progression, Christina J. Moss

USF Tampa Graduate Theses and Dissertations

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide with a low 5-year survival rate of only around 21%. This low 5-year survival rate is due to two main reasons. First, NSCLC is often diagnosed at the later stages when it has already metastasized. Second, NSCLC is an incredibly diverse, heterogenous disease making it very hard to target the true molecular oncogenic drivers. New targets for personalized therapeutics are needed based on the expression status of each individual lung cancer tumor.

One way of looking for these new therapeutics is to begin by identifying the oncogenotype …

A Health Evaluation Of Gulf Of Mexico Golden Tilefish (Lopholatilus Chamaeleonticeps) And Red Snapper (Lutjanus Campechanus) Following The Deepwater Horizon Oil Spill, Kristina Leigh Deak

USF Tampa Graduate Theses and Dissertations

A lack of baseline heath indices for offshore Gulf of Mexico (GoM) teleosts complicated impact assessments of the Deepwater Horizon (DWH) oil spill. While measurement of contaminant levels in fish after a pollution event can document exposure, such data fail to provide meaningful information about how this contact affects an animal's physiology. Controlled exposure studies have highlighted the utility of biomarkers that may indicate deleterious, long-lasting effects of pollutant exposure on various life stages of fish, however, their extrapolation to wild-caught, non-model species is challenging. In an increasingly chemically-saturated environment, it can also be difficult to separate the influence of …

Genome Maintenance Roles Of Polycomb Transcriptional Repressors Bmi1 And Rnf2, Anthony Richard Sanchez Iv

USF Tampa Graduate Theses and Dissertations

The coordination of transcription, replication, and DNA damage response (DDR) is vital for maintaining normal cellular homeostasis. All of these processes take place on the chromatin and thus, the temporal and spatial separation of the factors responsible are necessary for each to be correctly completed. Here we detail several novel processes contributing to this network.

BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1) which plays a key role in maintaining epigenetic silencing programs during development. Recently, BMI1 and other members of PRC1 like RNF2 have been implicated gene silencing during the DDR; however, the mechanism through which …

Mechanistic And Translational Studies On Skeletal Malignancies, Jeremy Mcguire

USF Tampa Graduate Theses and Dissertations

New treatment strategies are desperately needed for treating skeletal malignancy. Skeletal malignancies can be either primary cancer that originated in the bone, such as osteosarcoma, or metastatic cancer that spread from another organ to the skeleton, as in the case of breast or prostate cancer. In this thesis, I will detail two projects that focus on the discovery of new treatment strategies for both primary skeletal malignancy and metastatic skeletal malignancy.

The first project focuses on the primary skeletal malignancy, osteosarcoma, a rare cancer that is commonly diagnosed in children and young adults and metastasizes to the lungs. The survival …

A Novel Intramolecular Interaction In P53, Fan He

USF Tampa Graduate Theses and Dissertations

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. The activation process involves post-translational modifications that suppress p53 degradation by MDM2 and increase p53 DNA binding affinity. p53 is mutated in ~50% of human tumors, with higher frequency in specific tumor types and after relapse. Mutated p53 loses transcriptional activity and gains new functions that drive tumor progression. Both N-terminus (NT) and C-terminus (CT) of p53 contain intrinsically disordered regions. The p53 CT has well-documented effects in regulating DNA binding. CT truncated p53 mutants showed defective DNA binding and …

To Mid-Cell And Beyond: Characterizing The Roles Of Gpsb And Ypsa In Cell Division Regulation In Gram-Positive Bacteria, Robert S. Brzozowski

USF Tampa Graduate Theses and Dissertations

The bacterial cell division protein FtsZ is a tubulin homolog that forms a ring-like structure at the site of cell division in most bacterial species. There it acts as a scaffold, aiding in the recruitment of other divisome proteins to the site of cell division. Furthermore, studies focusing on the role of FtsZ treadmilling and septal peptidoglycan synthesis implicates that FtsZ plays a direct role in the ultimate closure of the division septum. Thus, many studies in the field of bacterial cell division have focused on FtsZ in terms of its spatial and temporal regulation as well as its ability …