Biochemistry, Biophysics, and Structural Biology Commons^™

The Mitochondrial Calcium Uniporter Regulator 1 (Mcur1) Matrix Domain Is A Self-Associated Multimer Sensitive To Divalent Cations, Naveed Siddiqui

Electronic Thesis and Dissertation Repository

Mitochondria are primarily appreciated for the generation of adenosine triphosphate (ATP), a chemical store of energy required by all cells. These organelles, however, also play key roles in apoptosis, autophagy and shaping cytosolic calcium (Ca²⁺) signaling via Ca²⁺ uptake into the mitochondrial matrix. This Ca²⁺ uptake is mediated chiefly via the mitochondrial Ca²⁺ uniporter (MCU), an inner mitochondrial membrane protein that oligomerizes to form a Ca²⁺ selective pore. MCU is regulated by several protein binding partners, including the recently identified MCU regulator-1 (MCUR1). MCUR1 stabilizes a higher order MCU heterocomplex through interactions with MCU …

Delineation Of New Mechanisms Of Dna Double Strand Break Repair, Songli Zhu

Theses & Dissertations

DNA damage is frequently induced in cells by both endogenous and exogenous agents. DNA damage, particular double strand breaks (DSBs) may lead to genomic instability, and the progression of cancer, aging, neurodegeneration, and other human diseases. The cell employs two major DSB repair pathways, including homologous recombination (HR) and Non-homologous end joining (NHEJ), but the detailed mechanisms of DSB repair remain to be further revealed.

In the first part of this study, we characterized a plasmid-based assay to investigate NHEJ repair in Xenopus egg extracts. Our data argued for a preference for the precise repair by the NHEJ machinery and …

Intra- And Inter-Molecular Signaling In A Cardiac Connexin: Role Of Cytoplasmic Domain Dimerization And Phosphorylation, Andrew J. Trease

Theses & Dissertations

As critical mediators of cell-to-cell communication, gap junctions (GJs) are comprised of membrane channels that directly link the cytoplasm of adjacent coupled cells thereby allowing for the passage of ions, small metabolites, and secondary messengers. Each channel is formed by the apposition of two connexons from adjacent cells, each composed of six connexin (Cx) proteins. Each GJ channel functions to promote signal propagation and synchronization of cells and tissues in organs. Furthermore, GJs are essential for proper propagation of cardiac action potentials from one cell to the next, leading to the coordinated contraction and relaxation of heart muscle powering circulation. …

Investigating Autophagy Dysfunction Induced By A Parkinson's Disease-Causing Mutation In Vps35, Abir Ashfakur Rahman

Boise State University Theses and Dissertations

Parkinson’s Disease (PD) is an idiopathic disorder with no known cure. With number of cases steadily rising around the world, it is imperative to turn to the underlying cellular and molecular mechanisms of the disease manifestation and neurodegeneration to craft novel modes of therapy. VPS35 is one of the few genes that have identified and definitively linked to familial PD. The particular mutation that has been associated is known to cause dysfunction of a key cellular process known as autophagy. This process is primarily responsible for clearance of unwanted, damaged or misfolded proteins, among other things. Our study reveals an …

Role Of P300 Zz Domain In Chromatin Association And Histone Acetylation, Yongming Xue

Dissertations & Theses (Open Access)

Transcription is strictly regulated by numerous factors including transcription coactivators. The p300 protein and its close paralogue CREB-binding protein (CREBBP, aka CBP) are well-known transcriptional coactivators that have intrinsic lysine acetyltransferase activity. The functions of p300/CBP largely rely on their capabilities to bind to chromatin and to acetylate the histone substrates. However, the molecular mechanisms underlying the regulation of these processes are not fully understood.

Through combination of various biochemical, biophysical and molecular approaches, we show that the ZZ-type zinc finger (ZZ) domain of p300 functions as a histone reader that specifically binds the N-terminal tail of histone H3. Crystal …

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, …

Fatty Acid Amides And Their Biosynthetic Enzymes Found In Insect Model Systems, Ryan L. Anderson

USF Tampa Graduate Theses and Dissertations

A fatty acid amide is precisely as the name suggests: A fatty acid (CHn-COOH), in which the hydroxyl group of the carboxylic acid is displaced by an amine functional group from a biogenic amine (R-NH2), ultimately forming an amide bond. Furthermore, these fatty acid amides can be composed of a variety of different acyl chain lengths donated by the fatty acid and a myriad of different biogenic amines. Thus, these molecules can be subdivided in a number of different ways including the separation of short chain (acetyl to heptanoyl) and long chain (palmitoyl to arachidonoyl) and also based off the …

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, …

Identifying Functional Components Of The Endoplasmic Reticulum Quality Control And Degradation Factor Edem1, Lydia Lamriben

Doctoral Dissertations

The ER Degradation-Enhancing Mannosidase-Like protein 1 (EDEM1) is a critical endoplasmic reticulum (ER) quality control factor involved in identifying and directing non-native proteins to the ER-associated protein degradation (ERAD) pathway. However, its recognition and binding properties have remained enigmatic since its discovery. Here we provide evidence for an additional redox-sensitive interaction between EDEM1 and Z/NHK that requires the presence of the single Cys on the α-1 antitrypsin ERAD clients. Moreover, this Cys-dependent interaction is necessary when the proteins are isolated under stringent detergent conditions, ones in which only strong covalent interactions can be sustained. This interaction is inherent to the …

The Interplay Between Polarity Regulators, Calcium, And The Actin Cytoskeleton During Tip Growth, Carlisle Bascom Jr

Doctoral Dissertations

Plant cell growth is a meticulously regulated process whereby the cell wall is selectively loosened to allow for turgor-pressure driven expansion. The rate of expansion must equal delivery of new material, or the cell will lyse. In many plant cells, this process happens diffusely around the cell. However, a number of plant cells have anisotropic shapes that require exquisite spatial control of secretion. One simple example of anisotropic patterning is tip growth; highly polarized cell expansion utilized by pollen tubes, root hairs, and moss protonemata. Investigating the role various molecules have in tip growth sheds light on how plant cells …

Effects Of Maternal Protein Restriction On The Pulmonary Surfactant System During The Early Life And Adulthood, Reza Khazaee

Electronic Thesis and Dissertation Repository

Fetal growth restriction (FGR) is defined by low birth weight and contributes to a variety of adult-onset diseases with different severities between males and females. However, the effects of FGR on the pulmonary surfactant are not fully elucidated. In this thesis, first, we investigated the FGR effects on the lung function and the surfactant system at the early postnatal life. It was hypothesized that FGR contributes to alterations of lung mechanics and the surfactant system during the neonatal period. Second, we assessed the FGR effects on the surfactant system in response to sepsis in adulthood. It was hypothesized that FGR …

Phospholipase D-Dependent Mtorc1 Activation By Glutamine, Elyssa Bernfeld

Dissertations, Theses, and Capstone Projects

Glutamine, the conditionally essential amino acid and most abundant amino acid in human sera, is a key nutrient required for sustaining cell proliferation. Glutamine is essential for nucleotide, protein, and lipid synthesis, all of which are essential for cell proliferation. The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex that acts as a sensor of nutrients, relaying signals for the shift from catabolic to anabolic metabolism. While glutamine plays an important role in activating mTORC1, the mechanism is not completely clear. Here we describe a Rag-independent mechanism of mTORC1 activation by glutamine that is dependent …

Renal Risk Variants Of Apolipoprotein L-1 Form Channels At The Plasma Membrane That Lead To A Cytotoxic Influx Of Calcium, Joseph A. Giovinazzo

Dissertations, Theses, and Capstone Projects

Apolipoprotein L-1 (APOL1) is a secreted protein that provides protection against several protozoan parasites due to its channel forming properties. Recently evolved variants, G1 and G2, increase kidney disease risk when present in two copies. In mammalian cells, overexpression of G1 and G2, but not wild-type G0, leads to swelling and eventual lysis. However, the mechanism of cell death remains elusive with multiple pathways being invoked, such as autophagic cell death mediated by a BH3 domain in APOL1, which we evaluated in this study. We hypothesized that the common trigger for these pathways is the APOL1 cation channel, which is …

Mechanisms Adopted By Dengue-2 Viruses To Induce Autophagy In Mammalian Cells, Sounak Ghosh Roy

Dissertations, Theses, and Capstone Projects

Dengue, the most rapidly spreading flavivirus, threatens to affect almost half of the human global population. We previously showed that dengue-2 protects canine kidney cells (MDCK) from cytotoxic chemicals. We showed, independently, that cell protection, as well as viral replication and maturation, are positively regulated by PI3K-dependent autophagy. However, we had not identified the specific pathway that induces autophagy in infected cells. The current study explores the role of a specific branch of the endoplasmic reticulum (ER) stress-mediated Unfolded Protein Response (UPR), the PERK/eIF2α/ATF4 pathway in the induction of autophagy by …

Direct Quantification Of Deubiquitinating Enzyme Activity In Single Intact Cells, Nora Safabakhsh

LSU Doctoral Dissertations

Challenges in drug efficacy occur during the treatment of most types of cancer due to the heterogeneity of the tumor microenvironment. This has led to the development of personalized medicine. Due to the clinical success of the proteasome inhibitors Bortezomib and Carfilzomib in treatment of multiple myeloma, interest has shifted towards molecularly-targeted chemotherapeutics for ubiquitin-proteasome system (UPS). Deubiquitinating enzymes (DUBs) are an essential part of this pathway which have been found to promote Bortezomib resistance in multiple myeloma patients. Unfortunately, there is a lack of specific, high throughput biochemical assays to characterize DUB activity in patient samples before and after …

Effect Of Arsenic Exposure On Early Eye Development In Zebrafish (Danio Rerio), Remy S. Babich

Electronic Theses and Dissertations

Arsenic is a metalloid that contaminates drinking water supplies worldwide. Due to concerns for human health, the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) have established a safe level in drinking water of ≤ 10 ppb. Arsenic has been shown to have carcinogenic effects in humans at high and low doses. Chronic exposure may result in dermal conditions such as hyperkeratosis and hyperpigmentation. Recently, arsenic exposure has also been linked to lower IQ values in children. The effect of arsenic on neurogenesis, specifically eye development, has not been widely explored. This study aimed to examine the …

The Role Of Mesenchymal Stromal Cells And Classical Dendritic Cells In The Maintenance And Regulation Of The Bone Marrow Niche, Jingzhu Zhang

Arts & Sciences Electronic Theses and Dissertations

The bone marrow niche is an important microenvironment for the regulation of normal and malignant hematopoiesis. The first discovered niche component is mesenchymal stromal cells, which are the major source for the production and secretion of multiple niche factors. Mesenchymal stromal cells are heterogeneous and various transgenes have been used to target non-identical but overlapping subpopulations. To further characterize the heterogeneity of mesenchymal stromal cells, we tested the targeting specificity of three tissue-specific Cre-recombinase transgenes. We show that in addition to osteoblasts, Ocn-Cre targets a majority of Cxcl12-abundant reticular (CAR) cells and arteriolar pericytes. Surprisingly, Dmp1-Cre also targets a subset …

The Regulation Of Notch Signaling By Src Kinase And Polyphenolic Compounds, Bryce David Lafoya

Boise State University Theses and Dissertations

Cellular signaling pathways provide cells with the means to sense their environment and communicate with other cells. The Notch signaling pathway is comprised of a set of protein machines which work in unison to coordinate cellular processes in response to stimuli coming from neighboring cells and changing microenvironmental conditions. Notch signaling is an important mode of cellular communication which is crucial to many processes involved in development and disease. During Notch activation, information about the extracellular environment is fed into the cell and relayed to the nucleus through a number of biochemical processes. The information-rich messages carried by Notch signaling …

Deciphering The Role Of Human Arylamine N-Acetyltransferase 1 (Nat1) In Breast Cancer Cell Metabolism Using A Systems Biology Approach., Samantha Marie Carlisle

Electronic Theses and Dissertations

Background: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can additionally hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. NAT1 expression varies inter-individually and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. Additionally, multiple studies have shown the knockdown of NAT1, by both small molecule inhibition and siRNA methods, in breast cancer cells leads to decreased invasive ability and proliferation and decreased anchorage-independent colony formation. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. Additionally, consequences …

The Role Of Developmental Timing Regulators In Progenitor Proliferation And Cell Fate Specification During Mammalian Neurogenesis, Jennifer S. Romer-Seibert

Graduate School of Biomedical Sciences Theses and Dissertations

Developmental timing is a key aspect of tissue and organ formation in which distinct cell types are generated through a series of steps from common progenitors. These progenitors undergo specific changes in gene expression that signifies both a distinct progenitor type and developmental time point that thereby specifies a particular cell fate at that stage of development. The nervous system is an important setting for understanding developmental timing because different cell types are produced in a certain order and the switch from stem cells to progenitors requires precise timing and regulation. Notable examples of such regulatory molecules include the RNA-binding …

Inhibition Of Ribosome Biogenesis Through Genetic And Chemical Approaches, Leonid Anikin

Graduate School of Biomedical Sciences Theses and Dissertations

In order to maintain the ability to generate proteins, proliferating cells must continuously generate ribosomes, designating up to 80% of their energy to ribosome biogenesis (RBG). RBG involves transcription of rDNA by RNA polymerases I (Pol I) and III (Pol III), expression of approximately 80 ribosomal proteins, and assembly of these components in a process referred to as ribosome maturation. During maturation, the Pol I transcribed 47S pre-rRNA undergoes a number of processing events, while simultaneously interacting with processing factors and ribosomal proteins that drive pre-ribosome assembly. Inhibition of RBG has become one of the pursued targets for cancer therapy …

Insight Into Translational Activation In Yeast Mitochondria, Julia Lynn Jones

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondrial function depends on over a thousand proteins, of which the majority are nuclear DNA-encoded and approximately one percent are mitochondrial DNA-encoded. The mitochondrial DNA of Saccharomyces cerevisiae contains eight protein-encoding genes, seven of which are required for proper function of the respiratory complexes and one encodes a ribosomal protein. The bigenomic nature of the oxidative phosphorylation complexes requires coordinated expression and regulation from both the nuclear and the mitochondrial genomes. It is currently unclear how this regulatory network operates. However, it is thought that nuclear genome-encoded messengers localized to the mitochondria aid in this coordination.

A family of proteins …

Characterization Of A Variant Of Tuberous Sclerosis Complex 2 And Its Interaction With Rheb, Sowmya Sivakumar

Graduate Theses and Dissertations

Protein-protein interactions are vital in maintaining proper function and homeostasis in cells. Some signaling pathways are regulated by G-proteins that work like switches to activate and deactivate pathways. Mutations in these proteins, their effectors or the interaction between proteins may cause dysregulation of signals that can lead to many diseases.

Rheb, Ras homology enriched in brain, is a Ras family GTPase that is vital in regulation of the mTOR (mammalian target of rapamycin) pathway that signals cell proliferation and growth. Due to the low intrinsic GTPase activity of Rheb, a GTPase activating protein (GAP), Tuberous Sclerosis Complex 2 (TSC2) down …

Egfr Signaling From The Early Endosome., Julie A. Gosney

Electronic Theses and Dissertations

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. When activated by a ligand at the plasma membrane, EGFR dimerizes with another ErbB family receptor, leading to kinase domain activation and transphosphorylation of C-terminus tyrosine residues. These phosphotyrosines act as crucial regulators of EGFR signaling as effector proteins dock to the receptor at these sites. The receptor undergoes clathrin-mediated endocytosis into early endosomes, where it can then be trafficked to a lysosome for degradation. However, the kinase domain of EGFR retains its activity during trafficking, suggesting that EGFR can continue …

Chronic Cadmium Exposure Alters Erα Dependency And Drug Sensitivity Of Breast Cancer Cells, Mathew Bloomfield

Dissertations, Masters Theses, Capstones, and Culminating Projects

The global prevalence of breast cancer in women illustrates the importance of identifying factors that contribute to disease onset and progression. Endogenous and environmental agents that interact with estrogen receptor alpha (ERα) have been shown to play a role in breast cancer etiology. Evidence from epidemiological studies and animal models has suggested that cadmium, a heavy metal that can activate ERα, contributes to the development and progression of breast cancer. Additionally, our lab showed that chronic cadmium exposure altered the expression of several ERα-responsive genes and increased the malignancy of MCF7 breast cancer cells. Although these studies support cadmium’s function …

Fluorescently Labeled Sirnas And Their Theranostic Applications In Cancer Gene Therapy, Stephen David Kozuch

Seton Hall University Dissertations and Theses (ETDs)

Gene therapy has emerged as a promising precision nano-medicine strategy in the treatment of numerous diseases including cancer. At the forefront of its utility are the applications of short-interfering RNA (siRNA), that silence oncogenic mRNA expression leading to cancer cell death through the RNA interference (RNAi) pathway. Despite the therapeutic potential, siRNAs are limited by poor pharmacological properties, which has hindered their translation into the clinic. Recent studies, however, have highlighted the applications of modified siRNAs, including the use of fluorescent probes and siRNA nanostructures in cancer detection and treatment. The siRNAs reported in this thesis are designed to target …

The Rational Design, Synthesis, Characterization, And Biological Evaluation Of Cancer-Targeting Immunostimulatory Peptide-Protein Conjugates And Tripeptides, Keith Smith

Seton Hall University Dissertations and Theses (ETDs)

With the advent of cancer immunotherapy and the rise in applications of synthetic biologics, there has been a steady decline in the incidence of cancer. Despite this trend, there is an anticipated 1.7 million new cancer cases with an estimated 610,000 deaths expected by the end of 2018.2 Therefore, the call for continued efforts in creating more effective treatment options are still in high demand. In this thesis, the rational design of a semi-synthetic cancer-targeting immunostimulatory peptide-protein bioconjugate—using N-succinimidyl carbamate chemistry is described. This bio-orthogonal chemistry approach was used to conjugate the synthetic Pep42, cancer-targeting peptide (CTP) and the immunostimulatory …

The Role Of Rad4 In Dna Repair And Its Interplay With Telomeres In Tetrahymena Thermophila, Emily Nischwitz

MSU Graduate Theses

Telomeres are repetitive parts of the genome that act as a protective end cap to the chromosomes. Telomeres are critical to the integrity and stability of the genome, therefore, ensuring that their sequence is maintained, even after damage, is crucial. Much of the pioneering work responsible for explaining telomeres has been conducted in ciliates, specifically in Tetrahymena thermophila. Telomeres in T. thermophila have a high amount of tandem thymine repeats (GGGGTT) and, thus, are susceptible to ultraviolet light (UV) induced lesions called pyrimidine dimers, which must be repaired by nucleotide excision repair (NER). In humans, Xeroderma Pigmentosum C (XPC) …

Computational Analysis Of Genomic Variants Affecting Predicted Microrna:Target Interactions In Prostate Cancer., Angélica Paola Hernández Pérez

KGI Theses and Dissertations

Prostate cancer (PCa) is the most common cancer of men in the United States and is third only to lung and colon as a cause of cancer death. Clinical behavior of the disease is variable and the combination of prostate-specific antigen (PSA) screening and Gleason score staging are currently the best available molecular and pathology tools to predict outcomes. Cancer biology research establishes microRNAs (miRNAs) as key molecular components in both normal and pathological states. Thus, elucidating miRNAs perturbed by genomic alterations will expand our understanding of the molecular taxonomy of PCa with the aim to complement current practices in …

Characterization Of She1 Spindle Role Using Ceullular, Biochemical, And Biophysical Methods, Yili Zhu

Doctoral Dissertations

During development, metaphase spindles undergo large movement and/or rotation to determine the cell division axis. While it has been shown that spindle translocation is achieved by astral microtubules pulling and/or pushing the cortex, how metaphase spindle stability is maintained during translocation remains not fully understood. In budding yeast, our lab has previously proposed a model for spindle orientation wherein the mitotic spindle protein She1 promotes spindle translocation across the bud neck by polarizing cortical dynein pulling activity on the astral microtubules. Intriguingly, She1 exhibits dominant spindle localization throughout the cell cycle. However, whether She1 has any additional role on the …