Molecular Biology Commons^™

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. …

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, …

Superoxide Dismutase C Modulates Macropinocytosis And Phagocytosis In Dictyostelium Discoideum, Cong Gu

FIU Electronic Theses and Dissertations

Macropinocytosis and phagocytosis, two actin-dependent and clathrin independent events of endocytosis, enable the cells such as macrophages and neutrophils to either internalize pathogens and initiates the human innate immune response or serve as a direct entry route for productive infection of pathogen. Dictyostelium discoideum, soil-living amoeba, a unicellular eukaryote that could professionally internalize fluid phase or particles several folds more than that of macrophages and neutrophils. Additionally, multiple key signaling pathways are conserved between Dictyostelium and mammalian cells, including pathways affecting small GTPases Ras and Rac and their downstream effectors, and F-Actin remodeling. All these traits makes Dictyostelium an …

Combined High-Speed Single Particle Tracking Of Membrane Proteins And Super-Resolution Of Membrane-Associated Structures, Hanieh Mazloom Farsibaf, Keith A. Lidke

Shared Knowledge Conference

Many experiments have shown that the diffusive motion of lipids and membrane proteins are slower on the cell surface than those in artificial lipid bilayers or blebs. One hypothesis that may partially explain this mystery is the effect of the cytoskeleton structures on the protein dynamics. A model proposed by Kusumi [1] is the Fence-Picket Model which describes the cell membrane as a set of compartment regions, each ~ 10 to 200 nm in size, created by direct or indirect interaction of lipids and proteins with actin filaments just below the membrane. To test this hypothesis, we have assembled a …

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 …

Iron-Dependent Cleavage Of Ribosomal Rna During Oxidative Stress In The Yeast Saccharomyces Cerevisiae, Jessica A Zinskie, Arnab Ghosh, Brandon M Trainor, Daniel Shedlovskiy, Dimitri G Pestov, Natalia Shcherbik

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

Stress-induced strand breaks in rRNA have been observed in many organisms, but the mechanisms by which they originate are not well-understood. Here we show that a chemical rather than an enzymatic mechanism initiates rRNA cleavages during oxidative stress in yeast (Saccharomyces cerevisiae). We used cells lacking the mitochondrial glutaredoxin Grx5 to demonstrate that oxidant-induced cleavage formation in 25S rRNA correlates with intracellular iron levels. Sequestering free iron by chemical or genetic means decreased the extent of rRNA degradation and relieved the hypersensitivity of grx5Δ cells to the oxidants. Importantly, subjecting purified ribosomes to an in vitro iron/ascorbate …

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 …

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 …

N-Terminal Domain Of Human Uracil Dna Glycosylase (Hung2) Promotes Targeting To Uracil Sites Adjacent To Ssdna-Dsdna Junctions, Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T Stivers

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias …

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 …

Structural And Functional Characterization Of Hyper-Phosphorylated Grk5 Protein Expressed From E. Coli, Joseph M. Krampen, John Tesmer, Qiuyan Chen

The Summer Undergraduate Research Fellowship (SURF) Symposium

G protein-coupled receptor (GPCR) kinases (GRKs) are proteins in the cell responsible for regulating GPCRs located on the cell membrane. GRKs regulate active GPCRs by phosphorylating them at certain sites which causes them to stop normal signaling on the membrane. This ultimately affects how the cell responds to its environment. GRK5 is a kinase of particular interest due to its involvement in the pathology of diseases such as cardiac failure, cancers, and diabetes. Understanding the structure and function of GRK5 is essential for discovering ways to manipulate its behavior with these diseases, but not much is known about how GRK5 …

Uncoupling The Folding And Binding Of An Intrinsically Disordered Protein, Anusha Poosapati, Emily Gregory, Wade M. Borcherds, Lucia B. Chemes, Gary Daughdrill

Molecular Biosciences Faculty Publications

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the …

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 …

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) …

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 …

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 …

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 …

Pias-Family Proteins Negatively Regulate Glis3 Transactivation Function Through Sumo Modification In Pancreatic Β Cells, Tyler M. Hoard, Xiaoping Yang, Anton M. Jetten, Gary T. Zeruth Dr.

Faculty & Staff Research and Creative Activity

Gli-similar 3 (Glis3) is Krüppel-like transcription factor associated with the transcriptional regulation of insulin. Mutations within the Glis3 locus have been implicated in a number of pathologies including diabetes mellitus and hypothyroidism. Despite its clinical significance, little is known about the proteins and posttranslational modifications that regulate Glis3 transcriptional activity. In this report, we demonstrate that the SUMO-pathway associated proteins, PIASy and Ubc9 are capable of regulating Glis3 transactivation function through a SUMO-dependent mechanism. We present evidence that SUMOylation of Glis3 by PIAS-family proteins occurs at two conserved lysine residues within the Glis3 N-terminus and modification of Glis3 by SUMO …

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 …

Examining Shsp-Substrate Capture And Chaperone Network Coordination Through Cross-Linking, Keith Ballard

Doctoral Dissertations

Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to protein misfolding diseases. They comprise a conserved core α-crystallin domain (ACD) flanked by an evolutionarily variable N-terminal domain (NTD) and semi-conserved C-terminal extension/domain (CTD). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that coordinate with ATP-dependent chaperones for refolding. To derive common features of sHSP-substrate recognition, I compared the chaperone activity and specific sHSP-substrate interaction sites for three different sHSPs from Arabidopsis (At17.6B), pea (Ps18.1) and wheat (Ta16.9), for which the atomic solution-state structures …

Anemarrhena Asphodeloides Bunge And Its Constituent Timosaponin‐Aiii Induce Cell Cycle Arrest And Apoptosis In Pancreatic Cancer Cells, Catherine B. Marelia, Arielle Sharp, Tiffany A. Shemwell, Y. C. Zhang, Brant R. Burkhardt

Molecular Biosciences Faculty Publications

Pancreatic cancer is one of the most recalcitrant and lethal of all cancers. We examined the effects of Anemarrhena asphodeloides (AA) and timosaponin‐AIII (TAIII), a steroidal saponin present in AA, on pancreatic cancer cell proliferation and aimed to elucidate their potential apoptotic mechanisms of action. Viability assays and cell cycle analysis revealed that both AA and TAIII significantly inhibited pancreatic cancer cell proliferation and cell cycle progression compared to treatment with gemcitabine, the standard chemotherapeutic agent for advanced pancreatic cancer. We identified a dose‐dependent increase in caspase‐dependent apoptosis and activation of pro‐apoptotic PI3K/Akt pathway proteins, with a subsequent downregulation of …

Exploring The Role Of Rna Polymerase Iii Complex Assembly On Ribosomal Dna Silencing In Saccharomyces Cerevisiae, Kyle Thomas Kern

LSU Doctoral Dissertations

The yeast rDNA region is host to a number of transcriptional regulatory elements, which work in conjunction to generate essential RNA subunits of ribosomes, as well as protecting the region from DNA damage. The role of RNA polymerase III complex binding at the 5S gene on rDNA silencing in the NTS2 region was investigated, both by use of a TY1:MET15 reporter insert and a MET15 gene integration at an endogenous SphI site. It was discovered that Pol III complexes do have an effect on reporter expression in the NTS2 region, though the specific effect was different based on the method …

Snf1 Cooperates With The Cwi Mapk Pathway To Mediate The Degradation Of Med13 Following Oxidative Stress, Stephen D Willis, David C Stieg, Kai Li Ong, Ravina Shah, Alexandra K. Strich, Julianne H Grose, Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

Eukaryotic cells, when faced with unfavorable environmental conditions, mount either pro-survival or pro-death programs. The conserved cyclin C-Cdk8 kinase plays a key role in this decision. Both are members of the Cdk8 kinase module that, along with Med12 and Med13, associate with the core Mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which releases cyclin C into the cytoplasm to promote mitochondrial fission and programmed cell death. The SCFGrr1 ubiquitin ligase mediates Med13 degradation dependent on the cell wall integrity pathway, MAPK Slt2. Here we show that the AMP kinase Snf1 activates a second …

Mapping Molecular Datasets Back To The Brain Regions They Are Extracted From: Remembering The Native Countries Of Hypothalamic Expatriates And Refugees, Arshad M. Khan, Alice H. Grant, Anais Martinez, Gully Apc Burns, Brendan S. Thatcher, Vishwanath T. Anekonda, Benjamin W. Thompson, Zachary S. Roberts, Daniel H. Moralejo, James E. Blevins

Arshad M. Khan, Ph.D.

Mathematical Modeling Of Nutrient Signaling And Growth In Saccharomyces Cerevisiae, Amogh P. Jalihal, Pavel Kraikivski, T.M. Murali, John J. Tyson

Biology and Medicine Through Mathematics Conference

No abstract provided.