Cell Biology Commons^™

A Transient Α-Helical Molecular Recognition Element In The Disordered N-Terminus Of The Sgs1 Helicase Is Critical For Chromosome Stability And Binding Of Top3/Rmi1, Jessica A. Kennedy, Gary W. Daughdrill, Kristina H. Schmidt

Molecular Biosciences Faculty Publications

The RecQ-like DNA helicase family is essential for the maintenance of genome stability in all organisms. Sgs1, a member of this family in Saccharomyces cerevisiae, regulates early and late steps of double-strand break repair by homologous recombination. Using nuclear magnetic resonance spectroscopy, we show that the N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix that extends from residue 25 to 38. Based on the residue-specific knowledge of transient secondary structure, we designed proline mutations to disrupt this α-helix and observed hypersensitivity to DNA damaging agents and increased frequency of genome rearrangements. In vitro binding assays …

Identification Of Proteins At Active, Stalled, And Collapsed Replication Forks Using Isolation Of Proteins On Nascent Dna (Ipond) Coupled With Mass Spectrometry, Bianca M. Sirbu, W. Hayes Mcdonald, Huzefa Dungrawala, Akosua Badu-Nkansah, Gina M. Kavanaugh, Yaoyi Chen, David L. Tabb, David Cortez

Molecular Biosciences Faculty Publications

Both DNA and chromatin need to be duplicated during each cell division cycle. Replication happens in the context of defects in the DNA template and other forms of replication stress that present challenges to both genetic and epigenetic inheritance. The replication machinery is highly regulated by replication stress responses to accomplish this goal. To identify important replication and stress response proteins, we combined isolation of proteins on nascent DNA (iPOND) with quantitative mass spectrometry. We identified 290 proteins enriched on newly replicated DNA at active, stalled, and collapsed replication forks. Approximately 16% of these proteins are known replication or DNA …

Engineering Microenvironments To Modulate Calcium Information Processing In Neuronal Cells, Kinsey Cotton Kelly

Doctoral Dissertations

Tissue engineered microenvironments were constructed to test the effects glial cells have on calcium information processing, and to mimic conditions in vivo for tumor invasion and residual cancer after resection of tumor. Submaximal, nM, glutamate (GLU) stimuli were applied to the engineered environments, and the resulting calcium dynamic behavior of neuronal cells was measured to help predict and interpret chaotic systems in the experimental realm. Calcium is a key signaling ion which signals through the N-methyl-D-aspartate (NMDA) glutamate receptor on the neuronal membrane. GLU binding to the NMDA receptor (NMDAR) causes a large and dynamic increase in neuronal intracellular calcium. …

Changes In Cell Morphology And The Cellular Localization Of Protein Kinase Dsk1 In Schizosaccharomyces Pombe In Response To Butylated Hydroxyanisole, Jacqueline T. Humphries

Scripps Senior Theses

Dsk1 is the Schizosaccharomyces pombe functional homolog of human SRPK1, an SR protein kinase that regulates localization and function of SR protein splicing factors involved in transcription, alternative splicing, and mRNA export. It has been shown that a Dsk1 deletion strain of S. pombe is sensitive to exposure to butylated hydroxyanisole (BHA), a phenol derivative commonly used as a food preservative. Little is known about how BHA interacts with cells on a functional level, although it has been shown to be cytotoxic and tumorigenic. The aims of this thesis are to study the effect of BHA on eukaryotic cells and …

Recruitment Of The Intracellular Ca2+ By Ultrashort Electric Stimuli: The Impact Of Pulse Duration, Iurii Semenov, Shu Xiao, Olga N. Pakhomova, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca2⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We …

A Path To Success? A Review Of Evolution, Development, And The Predictable Genome By David L. Stern, Hope Hollocher, Charles H. Pence, Grant Ramsey, Michelle M. Wirth

Faculty Publications

No abstract provided.

Interaction Between Centromeric Histone H3 Variant And Shugoshin, Visarut Buranasudja

Open Access Theses

Precise and faithful segregation of chromosome segregation during mitosis depends on the ability of the cell to regulate chromosome bi-orientation on the mitotic spindle. Shugoshin (Sgo1), the protector of meiotic centromeric cohesin, is required for proper establishment of chromosome bi-orientation. Sgo1 plays a crucial role as part of a mitotic tension sensor between sister chromatids. Recently, Sgo1 has been reported to interact with histone H3 at the pericentromere region, as an important factor for tension sensing and chromosome segregation. However, the role of Sgo1 in tension sensing at centromere is still elusive. The centromere is the region of attachment of …

Modulation Of Cell-Matrix Interaction For Cryopreservation Of Engineered Tissue, Angela Christine Seawright

Open Access Theses

Long term preservation of functional engineered tissues can significantly advance tissue engineering industry and regenerative medicine. Several preservation techniques have been proposed and investigated for this purpose, and cryopreservation is a leading candidate. While tissues are cryopreserved, ice forms in both the extracellular and intracellular spaces and causes freezing-induced spatiotemporal deformation of the tissue. During this process the cells undergo dehydration by the freezing-induced osmotic pressure difference and mechanical deformation, transmitted through cell-extracellular matrix adhesions. However, the significance and interaction of these cellular level transport and mechanics processes are not well understood. Therefore, this study aims to establish mechanistic understanding …

The Effects Of Hydrostatic Pressure On Early Endothelial Tubulogenic Processes, Ryan M. Underwood

Theses and Dissertations--Biomedical Engineering

The effects of mechanical forces on endothelial cell function and behavior are well documented, but have not been fully characterized. Specifically, fluid pressure has been shown to elicit physical and chemical responses known to be involved in the initiation and progression of endothelial cell-mediated vascularization. Central to the process of vascularization is the formation of tube-like structures. This process—tubulogenesis—is essential to both the physiological and pathological growth of tissues. Given the known effects of pressure on endothelial cells and its ubiquitous presence in the vasculature, we investigated pressure as a magnitude-dependent parameter for the regulation of endothelial tubulogenic activity. To …

Characterizing The Effects Of Glutaraldehyde On The Ryanodine Receptor Calcium Release Channel, Joshua Dov Strauss

Legacy Theses & Dissertations (2009 - 2024)

Ryanodine receptors (RyR) are large intracellular calcium release channels, which

Influence Of Mechanical Cues And The Extracellular Matrix On Cell Migration Patterns And The Proliferation Rates Of Cells, Tara Shane Alger

Wayne State University Theses

The mechanical environment of a cell and its tissue can impact multiple biological processes including development, wound healing, and metastasis. Specific cellular behaviors influenced by the mechanical microenvironment include differentiation, morphology, apoptosis, migration, and proliferation. In this thesis I have focused specifically on the effect of environmental stiffness and applied mechanical forces on cellular migration and proliferation, respectively. Using two different applications, both tailored to evaluate the mechanical forces alone on cellular behavior, I attempted to simulate the mechanical composition of the in vivo tissue microenvironments in vitro using polyacrylamide hydrogels. To test whether cells maintain a mechanical memory for …

Defining The Mechanism Of Enhanced Cellular Invasion Induced By Mechanical Stimulation, Snehal Sunil Ozarkar

Wayne State University Theses

Metastasis is a multistep process driven by various biochemical and mechanical factors, which eventually leads to formation of secondary tumors. The tumor mass is surrounded by basement membrane (BM) and stroma made of various extracellular matrix (ECM) proteins. During metastasis tumor cells disseminate from the primary tumor, breach the BM, invade the stroma, travel through blood and lymph and colonize tissues distant from the primary tumor. Formation of secondary tumors by metastasis is a leading cause of death in cancer patients. Even though plenty of research has been focused on biochemical factors affecting metastasis, information on role of mechanical factors …

Studies Of Regulated Exocytosis From Neuroendocrine Cells, Madhurima Das

Wayne State University Theses

In this thesis we study cargo release and fusion pore dilation during calcium triggered exocytosis and the co-localization of calcium sensing proteins essential for exocytosis, in neuroendocrine cells.

Pancreatic beta cells secrete several hormones, the most studied one being insulin. C-peptide is a protein which is co-stored with and secreted from the same vesicles as insulin. It is found in the soluble phase unlike insulin, which is found in the dense core. The pancreatic beta cells also secrete the Chromogranin B (CgB) which is mostly found in the dense cores of secretory vesicles. In chapter 1, we found that CgB, …

Optimization Of Labeling Techniques; Determination Of Best Parameter For Olfactory Mucosal Progenitor Cell Delivery And Study Of Effects Of Methylene Blue And Polyethylene Glycol In An Animal Model Of Spinal Cord Injury, Kiran Kumar Rokkappanavar

Wayne State University Theses

In the United States, the incidence of TSCI is about 40 per million persons per year, with approximately 250,000 living survivors of TSCI in the United States in July 2005. A number of human clinical trials (85) are ongoing using stem cells to evaluate the methods to reduce the injury following TBI. Among the stem cells, olfactory mucosal progenitor cells have several advantages with respect to ease of obtaining, fate of cells, etc. Our study was intended to optimize the labeling technique and to determine the best parameter to deliver the stem cells to the site of injury. We found …

Regulation Of Nuclear Localization Of The Sole Sumo-Conjugating Enzyme, Ubc9, Palak Sekhri

Wayne State University Theses

The covalent and reversible conjugation of small ubiquitin-like modifier (SUMO) proteins to hundreds of different cellular proteins is catalyzed by a cascade of enzymes including an E1-activating enzyme (SAE1/SAE2), an E2-conjugating enzyme (Ubc9) and multiple E3 ligases. As the only E2 enzyme for SUMO-conjugation, Ubc9 localizes mainly in the nucleus and plays an essential role in regulation of many cellular processes including cell cycle progression through mitosis, cell migration, genome stability, stress response, transcription, and nuclear transport in eukaryotic cells. It is hypothesized that the nuclear localization of Ubc9 is required for efficient sumoylation inside the nucleus because both the …

Attachment Ability And Melanoma Inhibitory Activity Mrna Expression Level Changes In Murine B16-F10 Melanoma Cells Post Nanosecond Electric Pulses, Hongxia Jia

Theses and Dissertations in Biomedical Sciences

The effects of high-voltage nanosecond electric pulses (nsEPs) on metastatic melanoma are still unclear. Hence, we applied one, two, three, and four 300 ns 40 kV/cm pulses to murine B16-F10 melanoma cells. Cell attachment ability was determined by comparing the number of floating cells and the percentage of attached cells. Melanoma inhibitory activity (MIA) is a secretory protein that is highly correlated with the malignancy and metastasis of malignant melanomas. We used MIA as our target to evaluate the effect of nsEPs on metastasis. Pulsed (experimental) and unpulsed (control) cells were incubated at 37°C under a 5% CO₂ atmosphere. …

Regulation Of The Tumor Suppresser P53 And Survivin By Ras And Ral Gtpases:Implications For Malignant Transformation, Awet G. Tecleab

USF Tampa Graduate Theses and Dissertations

Abstract

Although the critical role of the small GTPases Ras and Ral in oncogenesis has been well documented, much remains to be investigated about the molecular mechanism by which these GTPases regulate malignant transformation. The work under this thesis made two major contributions to this field. The first is the discovery that K-Ras, RalA and/or RalB are required for the maintenance of the high levels of the anti-apoptotic protein survivin in some human cancer cells, and the second is the demonstration that down regulation of K-Ras, RalA and/or RalB, but not Raf-1 or Akt1/2, stabilizes the tumor suppressor p53 and …

Phosphorylation Of Histone Deacetylase 6 Within Its C-Terminal Region By Extracellular Signal Regulated Kinase 1, Kendra Allana Williams

USF Tampa Graduate Theses and Dissertations

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Functional And Mutational Analysis Of Human Recq-Like Dna Helicases In Saccharomyces Cerevisiae, Hamed Mirzaei-Souderjani

USF Tampa Graduate Theses and Dissertations

RecQ-like proteins are a family of DNA helicases that are evolutionary conserved from prokaryotes to eukaryotes. A large amount of experimental evidence suggests these proteins have a major role in the maintenance of genome stability. In humans five RecQ like helicase have been identified (RecQL1, BLM, WRN, RecQL4, and RecQL5), three of which are associated with rare genetic disorders with sever chromosomal and developmental abnormalities, and an elevated predisposition to cancer. Among the disease associated RecQ-like helicases, BLM and WRN have been subject to extensive research, while our collective knowledge about the function of RecQL4 is still very limited. Similarly, …

Acidic Pericellular Ph: Effects On Proteolysis And Gene Expression As Determined In 3d Models Of Breast Carcinoma, Jennifer M. Rothberg

Wayne State University Dissertations

Among the non-cellular microenvironmental factors that contribute to malignancy of solid tumors is an acidic peritumoral pH. The first objective was to determine if an acidic extracellular pH observed in vivo (i.e., pHe 6.8) affects the activity of proteases, such as cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional cultures. At pHe 6.8 there were increases in pericellular active cysteine cathepsins and in degradation of DQ-collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of …