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Hydroxyurea Globally Inhibits Rna Synthesis, Destabilizes Mrnas, And Triggers Mec1-Opposed Rnapii Accumulation At 3’ Ends Of Genes, Pritpal Kaur

Theses and Dissertations

My dissertation includes two projects. The first project is focused on the regulation of glycolysis and oxidative metabolism by inducing heme synthesis in cancer cells. The second project deals with the effect of hydroxyurea on RNA synthesis. Heme is an essential cofactor for enzymes of the electron transport chain (ETC) and ATP synthesis in mitochondrial oxidative phosphorylation (OXPHOS). Heme also binds to and destabilizes Bach1, a transcription regulator that controls expression of several groups of genes important for glycolysis, ETC, and metastasis of cancer cells. Since many cancers are characterized by a high glycolytic rate regardless of oxygen availability, targeting …

Dna Damage Response Activates The Electron Transport Chain And Oxidative Metabolism By Two Parallel Mechanisms, Shreya Nagar

Theses and Dissertations

The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. Major part of DDR is coordinated by DNA damage checkpoint (DDC). In addition to DDC, eukaryotic cells also have DNA replication checkpoint (DRC) that is distinct from the DDC and specifically signals slowly progressing or arrested replication forks. DDR involves stalling or arrest of the cell cycle, initiation of DNA repair, and altered regulation of transcription, translation, and the ubiquitin-proteasome system. DDR also triggers transcription shut-off of histone genes. One of the key outcomes of DDC/DRC activation is the increased synthesis of the deoxyribonucleoside triphosphates (dNTPs), …

Mancozeb Exposure Results In Oxidative Stress And Disruption Of Mitochondrial Complexes Iii & Iv In Transformed Human Colon Cells, Amanda Dhaneshwar

Theses and Dissertations

The ethylene bisdithiocarbamate pesticides, a subclass of the dithiocarbamate pesticides, are broad spectrum organometallic fungicides used on a variety of crops. The fungicide Mancozeb is comprised of an ethylene bisdithiocarbamate backbone complexed to the metals manganese and zinc. Ingestion of food containing pesticide residue and occupational exposure are common routes of Mancozeb exposure. Previous work demonstrated that Mancozeb exposure to HT-29 colon cancer cells resulted in cytotoxicity, apoptosis, and disruption of mitochondrial complex I, II, and V activity. The current study investigated the effect of Mancozeb exposure on mitochondrial complex III and IV activity and oxidative stress parameters in the …

Interferon Gamma Signaling In Ovarian Cancer Cells, Bijaya Gaire

Theses and Dissertations

Interferon gamma (IFNγ) is a pleiotropic cytokine that can have, depending on the cellular and molecular context, both anti-tumor and pro-tumorigenic functions. IFNγ expression can be induced in cancer cells in response to radiation therapy or immune checkpoint blockade used in cancer treatment. We have previously shown that IFNγ induces expression of the Bcl3 proto-oncogene in ovarian cancer (OC) cells, resulting in their increased proliferation and migration, but the mechanisms are unknown. Here, I demonstrate that the IFNγ-induced Bcl3 expression is dependent on JAK1 and STAT1 signaling, and on p65 NFkB. Furthermore, my data show that the IFNγ-induced Bcl3 expression …

Gating And Pathogenic Mechanism Of Polycystin Ion Channels, Yan Wang

Theses and Dissertations

Autosomal dominant polycystic kidney disease (ADPKD), one of the most common and life-threatening human genetic diseases, is caused by mutations in PKD1 or PKD2 genes. PKD1 gene encodes PKD1 (also known as polycystin-1 or PC1) protein, which are the founding members of the PKD1 proteins family; while PKD2 encodes TRPP2 (also known as polycystin-2 or PC2) protein, the founding member of the TRPP subfamily. PKD1 family consists of PKD1, PKD1L1, PKD1L2, PKD1L3, and PKDREJ. Whereas the TRPP subfamily includes: TRPP2, TRPP3, and TRPP5. Proteins in PKD1 and TRPP families are collectively called polycystins. TRPP proteins can either form homotetrameric ion …

Genetically Encoded Iron Sensor Cell Line: A Model For Studying Ferroptosis And Cellular Iron Metabolism, Daoud F. Rahman

Theses and Dissertations

Cell death is a biological process that is essential in proper cellular homeostasis, development, and function. Malfunction of this system can lead to different diseases and cancers. Ferroptosis is a form of cell death that occurs as a result of the accumulation of lipid peroxides, that are dependent on intracellular levels of iron. Iron is essential to multiple metabolic processes in the cell, including the catalyzation of lipid peroxides. Thus, intracellular iron levels must be tightly regulated. Our goal is to create a cellular model to study and detect changes in intracellular iron levels. Through the use of a genetically …

Insight Into Eleutherodactylus Coqui Resilience And Anuran Hypoxia Tolerance Utilizing Pincho, Randy Ortiz

Theses and Dissertations

The onset of high-throughput RNA-sequencing (RNA-seq) technology allowed research into transcriptomics to accelerate exponentially aided by the rapid advancement of bioinformatic pipelines. The Pincho workflow, my first research endeavor, is a transcriptomic workflow developed to create an avenue of high-quality reconstructions from RNA-seq data. High-quality reconstruction standards entail longer transcripts, more complete transcripts, and more raw data utilization. We have discovered an ideal trio of assemblers between transABySS, rnaSPAdes and TransLiG that would best reconstruct next-generation sequencing data according to these standards. We utilized Pincho to drive two distinct experiments: (1) exploring the genetic basis for the successful invasion of …

Development Of Whole-Cell Biosensors For Early Detection Of Oral Squamous Cell Carcinoma, Sharona Adina Priyev

Theses and Dissertations

Accurate and timely diagnosis is crucial in preventing progression to advanced disease stages and in improving survival rates of oral squamous cell carcinoma (OSCC). Using synthetic biology tools, we aim to integrate synthetic biological circuits into the construction of a novel, whole-cell biosensor (WCB) that will detect salivary biomarkers of OSCC. In treating biological parts and genetic circuits as modular pieces, we can intentionally rearrange selected domains of interest to create new transcription factors that will enable the detection of molecules of interest. Our end-goal is the creation of portable devices carrying living cells that express our synthetic genetic circuits …

Design Of A Synthetic Temperature-Activated Epigenetic Biocontainment System, Owen Rivers Page

Theses and Dissertations

The need for an internal control to govern engineered strain survival is becoming apparent when considering the biosafety and ethics within biotechnological research. Using understudied regulatory elements known as RNA thermometers (RNATs) that can modulate protein translation based on temperature changes and a Type II restriction modification system, we posit the creation of a synthetic temperature-activated epigenetic biocontainment system, termed the ‘sentinel switch’. We have mined a wide variety of bacteria from Yersinia pestis to Synechocystis sp. looking for novel RNATs. A complementary synthetic biology approach based on library generation and screening of chimeric RNATs (ChRNATs) was also conducted. RNATs …

Identification Of Novel Phosphorylation Sites In Sars-Cov-2 Nucleocapsid Protein, Josie Daldegan Rezende

Theses and Dissertations

COVID-19, the 2019 disease outbreak, continues to cause severe mortality worldwide. Fast and reliable tests that identify the SARS-CoV-2 virus, which is responsible for causing the COVID-19 disease, are crucial for controlling the spread of the virus. Antigen tests are ideal for the rapid diagnosis of COVID-19. Current antigen tests for COVID-19 detect the SARS-CoV-2 nucleocapsid (N) protein. The nucleocapsid is the most abundant protein produced by the virus upon infection, which makes it a superior target for detection by an antigen test. Nucleocapsid proteins from several coronaviruses, including SARS, have previously been shown to be modified by posttranslational modification …

Regulation Of Interferon- Induced Interleukin-8 And Pd-L1 Expression In Ovarian Cancer Cells, Sveta Padmanabhan

Theses and Dissertations

Interferon-γ (IFNγ) is a pleiotropic cytokine that has a crucial role in immune response and tumor immunity. Because of its anti-tumor effects, IFNγ has been used in cancer treatment. However, IFNγ also has tumor-promoting functions that are less well understood. Here, I show that IFNγ induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) in ovarian cancer (OC) cells. The IFNγ-induced IL-8 expression is dependent on JAK1, STAT1, and p65 NFκB, and is associated with an increased occupancy of K314/ 315 acetylated p65 NFκB and Ser-727 phosphorylated STAT1 at the IL-8 promoter. Neutralization of IL-8 using anti-IL-8 antibody …

Functional Modulation Of The Cav3.1 Calcium Channel By Alternative Splicing At The C-Terminus, Ruizhi Wang

Theses and Dissertations

The CACNA1G gene encodes for Cav3.1, the alpha-1G subunit of a low-voltage-activated T-type calcium channel, which plays crucial roles in cardiac and smooth muscle cells and neurons by influencing the transmembrane potentials and regulating intracellular Ca2+ signaling. Heterozygous missense mutations in the CACNA1G gene cause spinocerebellar ataxia-42 (SCA42), a heterogeneous group of neurodegenerative disorders characterized by brainstem and cerebellum degeneration, and mice that are null for this gene present severe motor coordination defects, abnormal electrical conductance in the heart, aberrant action potentials in the brain, disrupted sleeping patterns, and absence epilepsy. Different splice variants of Cav3.1 have been described, and …

Dithiocarbamates Mancozeb And Disulfiram Induce Striated Muscle Myopathy And Neuropathy In Long-Evans Rats, Olivia Stephenson

Theses and Dissertations

Mancozeb (MZ), an ethylene bis-dithiocarbamate complexed with the metals manganese and zinc, is a broad-spectrum fungicide used primarily on orchards and vineyards. Disulfiram (DS, Antabuse®), a diethyldithiocarbamate, is an FDA-approved drug used for alcohol aversion therapy. Both dithiocarbamates (DTCs) have shown neurological effects including motor impairment, muscle weakness, and hind-limb paralysis, although the mechanism of toxicity is not understood. The purpose of this study is to determine how these neurotoxic compounds are damaging striated muscle of rats. Adult, male Long-Evans rats were administered either 250mg/kg MZ or 750mg/kg DS via oral gavage 3x/week for four weeks. Animals were euthanized 24 …

Evolution And Flower Shape From The Hawaiian Lobelioids To The Campanulaceae, Jingjing Tong

Theses and Dissertations

Campanulaceae, with roughly 2,400 species, is divided into five subfamilies, in part, by differences in their floral orientation and symmetry, with broad transitions between radial and bilateral floral symmetry. The Hawaiian lobelioids in the Campanulaceae is the largest endemic clade of Hawaiian angiosperms, and they have long been viewed as one of the most spectacular examples of an adaptive radiation in plants. In the first part of our study, we focus on Clermontia, which is one of the largest genera of Hawaiian lobelioids, employing variable low-copy nuclear gene sequences to determine their phylogenetic relationships. We use intron regions from four …

Alpha-Synuclein Multimerization Is Dependent On Structural Characteristics Of Repeated Ktkegv Regions, Benjamin Ira Rosen

Theses and Dissertations

Parkinson’s disease (PD) is the most common movement disorder and is characterized by neuronal loss and the presence of Lewy bodies in dopaminergic neurons of the substantia nigra pars compacta. PD is a chronic, progressive, and irreversible neurodegenerative disorder associated with the selective loss of dopaminergic neurons. Initially described in an Ayurvedic medical treatise and Galen’s writings, and later by James Parkinson in 1817, the most common symptoms of PD are resting tremors, abnormal posture and gait, and muscle rigidity. Approximately 1 million people are living with PD in the United States and worldwide estimates are between 7 and 10 …

Microrna Regulation And Cellular Proteostasis In Parkinson's Disease, Alberim Kurtishi

Theses and Dissertations

Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder predominately affecting the aging population. It is the second most common neurodegenerative disorder after Alzheimer’s disease (AD) affecting over 1 million individuals in the US alone. Recent studies have tried to understand the exact nature in which healthy neurons transition to a degenerative state in PD. There are a multitude of combinatory factors that can lead to the development of PD including environmental factors, genetic factors, and aging. The majority of PD cases are sporadic in nature, however familial cases account for 5-10% of total PD cases world-wide. The hallmark …

The Ion Channel Trpm7 Regulates Zinc Depletion-Induced Mdmx Degradation, Herui Wang

Theses and Dissertations

Zinc deficiency has been linked to human diseases including cancer. p53, a zinc-containing transcription factor, is the most frequently mutated tumor suppressor protein in human cancer. MDM2 and MDMX are zinc- containing proteins and crucial negative regulators of p53, which have been found to be amplified or overexpressed in various cancers. To investigate the effects of zinc depletion on the p53 signaling pathway, we have used ion chelators TPEN and bispicen as well as zinc-deficient medium to treat cells. We report here that zinc depletion results in MDMX degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc recovers …

Rna Splicing In Neuron Physiology And Neurodegenerative Diseases, Md Faruk Hossain

Theses and Dissertations

Gene expression is regulated at multiple levels, including transcription, RNA editing, pre-mRNA splicing, mRNA export, translation, and posttranslational modifications. Alternative splicing is a process by which exons can be included or excluded, giving rise to multiple mRNA isoforms from the same transcript. Alternative splicing is an important mechanism in developmental, tissue- and cell-specific control of gene expression, and it is key for expanding proteomic diversity and complexity from a limited number of genes. Moreover, more than 95% of multiexon genes undergo alternative splicing in humans, and about half of all disease-causing point mutations in humans affect pre-mRNA splicing, including neurological …

Characterizing Ferroptosis Pathways In In Vitro Motor Neuron Models, Alejandra Martinez

Theses and Dissertations

Ferroptosis is a non-apoptotic regulated form of cell death driven by the toxic accumulation of lipid peroxides and the presence of iron. The mechanism by which ferroptosis operates requires further investigation; here I will discuss the findings regarding the role of ferroptosis in different cellular models as well as the genes that may be involved with either promoting or hindering the ferroptotic pathway. NSC-34 is a hybrid cell line of neuroblastoma and primary spinal cord cells that can be differentiated into a motor neuron-like state allowing the characterization of ferroptosis in two distinct cellular conditions. Additionally, iNIL cells are a …

Boost The Dsicovery Of Mrp7/Abcc10 Substrates And Inhibitors: Establishment Of New In Vitro And In Silico Models, Jingquan Wang

Theses and Dissertations

ATP-binding cassette (ABC) transporters are responsible for the efflux of structurally distinct endo- and xenobiotics energized by ATP hydrolysis. MRP7/ABCC10 belongs to the 10th member of subfamily C and responsible for mediating MDR against a series of chemotherapeutic drugs such as taxanes, epothilones, Vinca alkaloids, anthracyclines and epipodophyllotoxins. Establishment of new in silico and in vitro models for MRP7 substrates/inhibitors prediction Considering the limited knowledge of MRP7, we established a homology model based on bovine MRP1 cryo-EM models. The final model was used for protein global motion analysis and docking analysis. Before docking, potential drug binding pockets were identified and …

Anticancer Effect Of Indanone-Based Thiazolyl Hydrazone Derivative On P53 Mutant Colon Cancer Cell Lines: In Vitro And In Vivo Study, Silpa Narayanan

Theses and Dissertations

Colorectal cancer is the third leading cause of cancer related deaths in the United States. Currently, irinotecan, a topoisomerase I inhibitor, is an important anticancer drug approved for patients with advanced or recurrent colorectal cancer. Considering the low response rate and the events of high toxicity caused by irinotecan, we evaluated a series of thirteen thiazolyl hydrazone derivatives of 1-indanone for their potential antineoplastic activity and four compounds showed promising anticancer activity against most of the tested colon cancer cell lines with IC50 values ranging from 0.41 ± 0.19 to 6.85 ± 1.44 μM. It is noteworthy that the compound, …

Design, Synthesis And Pharmacological Evaluation Of Quinazolinamine Derivatives As Bcrp And P-Gp Inhibitors With Improved Metabolic Stability, Chao-Yun Cai

Theses and Dissertations

A series of twenty-two quinazolinamine derivatives showing potent inhibitory activities on BCRP and P-gp was synthesized. The reversal study showed that when combined with the potent dual BCRP and P-gp inhibitors 7-8, 29-31, and 34, the IC50 value of mitoxantrone was decreased from 6.50 µM to the range of 0.24 - 0.35 µM for BCRP, and IC50 value of colchicine was decreased from 7.34 μM to the range of 0.12 - 0.29 µM for P-gp. Cyclopropyl quinazolinamine 29 (VKCY-1), which was a dual BCRP and P-gp inhibitor, and azide quinazolinamine 40 (VKCY-2), which was a BCRP inhibitor, were selected for …

Mechanism Of Dna Damage Checkpoint (Ddc)- Mediated Repression Of Histone Mrnas And Role Of Acetyl-Coa In Histone Transcription, Madhura Bhagwat

Theses and Dissertations

My dissertation contains two projects. The first project is focused on the mechanism of DNA damage checkpoint (DDC)- mediated repression of histone mRNAs. The second project deals with the role of acetyl-CoA in histone transcription. Proliferating cells coordinate histone and DNA synthesis to maintain correct stoichiometric amounts for chromatin assembly. Histone mRNA levels must be repressed when DNA replication is inhibited to prevent toxicity and genome instability due to free non-chromatinized histone proteins. In the first project we show that, unlike in mammalian cells, DNA replication stress does not trigger decay of histone mRNAs in the yeast Saccharomyces cerevisiae. However, …

Effect Of Inhibition Of Glycogen Catabolism In Hepatocellular Carcinoma Cells, Shrikant Barot

Theses and Dissertations

Metabolic reprogramming is one of the important features of cancers, and there has been growing interest in targeting metabolic proteins to treat cancer. Glycogen is a polymer of glucose and serves as its storage unit in cells. Glycogen can provide energy to cells during the situations of high energy demand. A number of tumors are known to contain high levels of glycogen than their normal tissue counterparts. The liver plays an essential role in maintaining glucose homeostasis in the body via storing it into glycogen. The significance of glycogen metabolism in patients suffering from hepatocellular carcinoma (HCC) has not been …

Investigating The Patterns And Mechanisms Of Alternative Splicing Of Cav1.3 In The Mouse., Samuel Sabzanov

Theses and Dissertations

AL-type voltage-gated calcium channel Cav1.3 contains a pore-forming α-1 subunit that is encoded by Cacna1d, a gene which has been shown to undergo alternative splicing (AS). Mutations in CACNA1D have been associated with neurological diseases such as spontaneous seizure disorder and epilepsy. Previous studies have shown that AS of the C-terminus of Cav1.3 results in a truncated variant of exon 42 (E42) that, when compared to the full-length exon, lowers the voltage threshold needed to activate the calcium channel. Therefore, AS of the a1-subunit can modulate the physiological properties of Cav1.3. As a lower voltage threshold of calcium channels increases …

Acetoacetate Enhances Oxidative Metabolism And Response To Toxicants Of Cultured Renal Cells, Trudi Maria Denoon

Theses and Dissertations

In vitro methods can be cost effective and facilitate large scale drug screening in the pharmaceutical industry. Renal proximal tubular cells have been a prime target for drug induced toxicity. LLC-PK1 cells possess many transport functions of the proximal tubule epithelia, however, its energy metabolism differs from the highly oxidative tubule metabolism in vivo. This may impact its reliability in predicting toxicity. Modulating the growth media composition has been shown to shift cultured liver and muscle cells from high glycolytic activity to increased oxidative metabolism, but few studies have been done on renal cells. This study is designed to test …