Molecular Biology Commons^™

Mechanisms Underlying The Heterogeneous Sensitivities Of Cancer Cells To Proteasome Inhibitors, Matthew C. White

Dissertations and Theses (Open Access)

The mechanisms underlying cellular response to proteasome inhibitors have not been clearly elucidated in solid tumor models. Evidence suggests that the ability of a cell to manage the amount of proteotoxic stress following proteasome inhibition dictates survival. In this study using the FDA-approved proteasome inhibitor bortezomib (Velcade®) in solid tumor cells, we demonstrated that perhaps the most critical response to proteasome inhibition is repression of global protein synthesis by phosphorylation of the eukaryotic initiation factor 2-α subunit (eIF2α). In a panel of 10 distinct human pancreatic cancer cells, we showed marked heterogeneity in the ability of cancer cells to induce …

Characterizing The Role Of Cortactin In Actin Pedestal Assembly By Enterohemorrhagic Escherichia Coli (Ehec), Sarah E. Grout

Honors Scholar Theses

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne cause of bloody diarrhea and renal failure. During colonization of the intestine, EHEC injects the transmembrane receptor protein Tir and the cytoplasmic effector protein EspFU into host cells to reorganize the actin cytoskeleton into adhesion “pedestals.” EspFU has been shown to bind and activate the actin nucleation factor N-WASP to drive actin polymerization into pedestals. However, EspFU can still assemble pedestals in cells lacking N-WASP, suggesting that this effector protein is able to also trigger N-WASP-independent pathways of actin polymerization during infection. Cortactin is an atypical nucleation factor that localizes to pedestals, …

Detection Of Viable Microorganisms Using Propidium Monoazide, Erik J. Mcfarland, Adrian Ponce Dr.

STAR Program Research Presentations

Propidium monoazide (PMA) is a molecular tool used to assess viability of microorganisms. Currently, PMA is thought to discern viability through membrane permeability; PMA enters only membrane compromised cells, irreversibly crosslinks to theirDNAand precipitates theDNAout of solution, preventing it from being amplified during polymerase chain reaction (PCR). Using PMA on a sample of live and dead microorganisms results in only theDNAof living organisms being amplified and identified. Therefore, a comparison ofPCRresults with and without PMA allows one to determine the live fraction and total population, respectively.

Current literature provides conflicting evidence as to the effectiveness of the technique. Our research …

Generation And Characterization Of Peptide Fusion Proteins, Brianna L. Probasco

Graduate School of Biomedical Sciences Theses and Dissertations

Pathogenic Th17 cells drive progression of many autoimmune diseases. Th17 cells develop from naïve T cells in the immune system after antigen-driven stimulation in a specific cytokine environment. Normally, T cells act to fight off infection, but when not properly controlled, they can cause disease. The cytokine interleukin-23 (IL-23) plays an essential role in the expansion of pathogenic Th17 cells. IL-23 is a heterodimeric protein, composed of a p19 alpha chain and a p40 beta chain. The p40 is also part of IL-12 and binds to the IL-12 receptor beta 1 (IL-12Rβ1) subunit. Thus, it follows that the IL-23 receptor …

Extracellular Proteases Are Key Mediators Of Staphylococcus Aureus Virulence Via The Global Modulation Of Virulence-Determinant Stability, Stacey L. Kolar, J. Antonio Ibarra, Frances E. Rivera, Joe M. Mootz, Jessica E. Davenport, Stanley M. Stevens Jr., Alexander R. Horswill, Lindsey N. Shaw

Molecular Biosciences Faculty Publications

Staphylococcus aureus is a highly virulent and successful pathogen that causes a diverse array of diseases. Recently, an increase of severe infections in healthy subjects has been observed, caused by community-associated methicillin-resistant S. aureus (CA-MRSA). The reason for enhanced CA-MRSA virulence is unclear; however, work suggests that it results from hypersecretion of agr-regulated toxins, including secreted proteases. In this study, we explore the contribution of exo-proteases to CA-MRSA pathogenesis using a mutant lacking all 10 enzymes. We show that they are required for growth in peptide-rich environments, serum, in the presence of antimicrobial peptides (AMPs), and in human blood. …

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 …

Regulation Of The Glycine Transporter 1 (Glyt1) By Pkca-Dependent Ubiquitination, Susana Barrera

Open Access Theses & Dissertations

Glycine is one of the major inhibitory neurotransmitters in the central nervous system. It is implicated in the regulation of motor-sensory function such as; pain perception, reflex responses, and is essential for early development of the CNS. In addition to its inhibitory actions, it acts as an obligatory co-agonist for the activation of glutamatergic NMDARs, which are of pivotal importance in the process of learning and memory, its dysfunction leads to several neurodegenerative diseases. Two high affinity Na+/Cl- dependent transporters GlyT1 and GlyT2 tightly regulate the availability of glycine present at the synapse for the activation of NMDARs and GlyRs. …

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 …

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 …

A Novel Fkbp52/Β-Catenin Complex Specifically Regulates Androgen Receptor Activity, Cheryl Lynne Storer

Open Access Theses & Dissertations

The androgen receptor complex plays an essential role in prostate cancer progression due to exploitation of the androgen receptor (AR) as a transcription factor. The final stage of the receptor complex consists of a dimerized receptor, a dimeric heat shock protein (Hsp90), the cochaperone p23, and an immunophilin. Hormone-dependent prostate cancer progresses due to key interactions between the androgen receptor complex and its ligand α dihydrotestosterone (DHT). While current treatments focus on blocking the androgen receptor-ligand interactions, these therapies are no longer effective in advanced stage, hormone-refractory prostate cancer (HRPC).

Therefore, we have been interested in targeting other members of …

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. …

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 …

The Transcriptional Regulation Of Flagellin-Induced Innate Protection Of The Cornea: Role Of Irf1 And Atf3, Gi Sang Yoon

Wayne State University Dissertations

Pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against infections by reprogramming gene expression. This study explored the flagellin-induced modifications of transcription factor expression and function, specifically of IRF1 and ATF3 in corneal epithelial cells to elucidate the transcriptional mechanisms underlying the protective function of flagellin on the cornea.

Initially we used Superarray to screen for transcription factors and identified Interferon Regulatory Factor (IRF) 1 and Activating Transcription Factor (ATF) 3 as the most drastically affected genes by flagellin pretreatment in P. aeruginosa challenged human corneal epithelial cells (CEC). However, flagellin pretreatment had opposite effects …

Characterization Of G-Patch Motif Contribution To Prp43 Function In The Pre-Messenger Rna Splicing And Ribosomal Rna Biogenesis Pathways, Daipayan Banerjee

Theses and Dissertations--Biology

The DExD/H-box protein Prp43 is essential for two biological processes: nucleoplasmic pre-mRNA splicing and nucleolar rRNA maturation. The biological basis for the temporal and spatial regulation of Prp43 remains elusive. The Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 G-patch proteins bind to and activate the Prp43 DExD/H box-helicase in pre-mRNA splicing (Spp382) and rRNA processing (Sqs1, Pxr1). These Prp43-interacting proteins each contain the G-patch domain, a conserved sequence of ~48 amino acids that includes 6 highly conserved glycine (G) residues. Five annotated G-patch proteins in baker’s yeast (i.e., Spp382, Pxr1, Spp2, Sqs1 and Ylr271) and with the possible exception of the uncharacterized Ylr271 …

Molecular Mechanisms Involved In Anhydrobiosis Of Insect Cells, Diyagama Arachchi Ralalage Dilini Sewwandi Samarajeewa

Masters Theses

Animals possessing tolerance to extreme water stress are termed anhydrobiotes. Many desiccation tolerant organisms respond to water stress by intracellular accumulation of selected sugars such as trehalose and larger macromolecules such as Late Embryogenesis Abundant (LEA) proteins and thereby maintain the cell viability. Evidence indicates that the presence of trehalose and Late Embryogenesis Abundant (LEA) proteins may work synergistically to confer cellular protection during drying in eukaryotic cells. We evaluated any increase in cellular desiccation tolerance by expressing different LEA proteins in a non-desiccation tolerant insect cell line Drosophila melanogaster (fruit fly) (Kc167 cells) in the presence of trehalose.

Transgenic …

Sirt1 Regulation Of The Heat Shock Response In An Hsf1-Dependent Manner And The Impact Of Caloric Restriction, Rachel Rene Raynes

USF Tampa Graduate Theses and Dissertations

The heat shock response (HSR) is the cell's molecular reaction to protein damaging stress and is critical in the management of denatured proteins. Activation of HSF1, the master transcriptional regulator of the HSR, results in the induction of molecular chaperones called heat shock proteins (HSPs). Transcription of hsp genes is promoted by the hyperphosphorylation of HSF1, while the attenuation of the HSR is regulated by a dual mechanism involving negative feedback inhibition from HSPs and acetylation at a critical lysine residue within the DNA binding domain of HSF1, which results in a loss of affinity for DNA. SIRT1 is a …

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 …

Epigenetic Modifiers To Augment The Immunogenicity Of Chronic Lymphocytic Leukemia, Jason A. Dubovsky

USF Tampa Graduate Theses and Dissertations

Cancer cells employ a litany of immunosuppressive and immunevasive strategies to avoid detection and elimination by the various arms of the innate and adaptive immune system. Many hematologic and solid tumors progressively develop a specialized microenvironment which promotes tissue restructuring inflammation while masking the immune signature of the tumor cells themselves. Chronic lymphocytic leukemia, a malignancy of mature B lymphocytes must constantly balance on the precipice of immune recognition. A mature antigen presenting cell themselves, CLL clonal growth is dependent on the very interactions which, if effective, could potentially lead to their demise. To circumvent this, CLL clones set up …

Toc Complex Formation: An Investigation Of The Interactions Governing Toc Complex Composition And Assembly, Steven R. Siman

Theses and Dissertations (Comprehensive)

Chloroplast-destined preproteins are translated in the cytosol, and posttranslationally targeted to and translocated across the double envelope membrane of the chloroplast by the coordinated activities of two translocon complexes: the Translocons at the Outer and Inner envelope membrane of the Chloroplast (TOC and TIC, respectively). In the model organism Arabidopsis thaliana the core TOC components include two families of GTPase receptors: TOC159 (atToc159, -132, and -120) and TOC34 (atToc33 and -34). These receptor families are hypothesized to assemble into distinct complexes and recognize transit peptides present on the N-terminus of chloroplast-destined preproteins. The GTPase domains of the TOC159 and TOC34 …

Molecular Mechanisms Regulating Neonatal Oocyte Survival And Primordial Follicle Formation In The Mouse Ovary, Robin L. Jones

Biology - Dissertations

In mammals, formation of the primordial follicle is a complex process involving the breakdown of germ cell cysts, where oocytes must separate from each other and subsequently become surrounded by somatic cells. As cysts separate, a large number of germ cells are lost by apoptosis, however the mechanisms by which cyst breakdown and germ cell death occur are not well understood. We first hypothesized that two anti-apoptotic regulators from the BCL2 family of proteins, BCL2 and MCL1, may be responsible for regulating neonatal oocyte survival. To elucidate the effects of BCL2 in the neonatal ovary, we examined ovaries of both …

An Analysis Of The Redox Properties And Stability Of Chlamydomonas Reinhardtii Cytochrome F, Cytochrome C6, And Mutants Thereof, Nicole Lynn Vanderbush

Graduate Theses and Dissertations

This body of work presents mutagenesis studies conducted on two c-type cytochromes from Chlamydomonas reinhardtii. Cytochrome f, a unique c-type cytochrome, is investigated in regards to its redox potential, the dependence of the redox potential, and the thermal stability of the protein. The mutations made were Y1F, Y9F, Y160F, Y160L, R156L, and R156K. The residues that were mutated surround the heme. It was found that, relative to the wild-type, only the Y160L and R156 mutants showed any difference in midpoint potential at pH 7. Wild-type and mutants both had a midpoint potential that was dependent upon pH indicating that none …

Identification Of Two Functional Sumo Interacting Motifs (Sims) In The Non-Structural Protein Of Influenza A Virus Ns1a, Lorellie M. Lewis, German Rosas-Acosta

COURI Symposium Abstracts, Summer 2012

We have previously shown that the non-structural protein of Influenza A virus NS1A is post-translationally modified by conjugation of the Small Ubiquitin-like MOdifier (SUMO). Our functional studies have also demonstrated that SUMO conjugation (SUMOylation) affects NS1A's ability to neutralize the cellular interferon response of the host cell and other functions associated to NS1A. Importantly, we have also shown that influenza infections trigger a global increase in cellular SUMOylation and have recently demonstrated that such an increase is mediated by NS1A. This observation led us to investigate the molecular mechanism endowing NS1A with the ability to increase global cellular SUMOylation. Sequence …

Dietary Analysis And Epigenetic Comparisons Of Drosphila Melanogaster Through Multiple Generations, Xu (Kevin) F. Zheng

Holster Scholar Projects

Nutrition and the lack thereof has been a demanding issue in both the undeveloped and developed country. The unbalance diet between healthy and unhealthy diet has led to many of the problems presented in society such as obesity, heart disorders, and loss of longevity. It is now known that calorie may not provide the most accurate information in terms of the lifestyle and the well-being of humans. Rather the composition of the calorie is what becomes integral in people's understanding of the effect of food physiologically. With this knowledge, we seek to use a Drosophila melanogaster as a model organism …

Host Pathogen Interactions: Is Arabidopsis Thaliana Remembered By Its Nemesis Pseudomonas Syringae?, Daniel Z. Kreiser

Lawrence University Honors Projects

Plants contain innate immune systems that deter pathogen infection. Pattern recognition receptors bind microbe-associated molecular patterns (MAMPs), triggering immunity. MAMPs are proteins exclusive to pathogens that are typically indispensable for their survival. For this reason, MAMPs cannot be mutated or removed without causing pathogen death. However, this does not necessitate constitutive expression of MAMPs. In this study, the MAMP response of Arabidopsis thaliana was utilized to determine differential detection of MAMPs expressed by Pseudomonas syringe pv. tomato DC3000 when pretreated with A. thaliana. Results demonstrated that more MAMPs are detected when P. syringae had previously encountered A. thaliana, …

Elucidating The Mechanism Of Antimigratory Activity Of Cardiac Glycosides, Joshua H. Johnson

Honors Scholar Theses

The focus of this research is on cell migration and how it can be better understood through the use of small molecules that modulate cell migratory activity. The results have particular relevance in the realm of cancer pharmacology. Cardiac glycosides, which are known inhibitors of the eukaryotic Na⁺/K⁺-ATPase, have been determined to have antimigratory activities through the screening of several small molecule libraries. Here we investigate the antimigratory activities of the cardiac glycoside digitoxin as well as its analogs that we synthesized. Antimigratory activity was determined by conducting a wound closure assay with MDA-MB-231 human breast …

Improvements To The Forensic Analysis Of Mitochondrial Dna Typing, Elizabeth Montano

Honors Scholar Theses

Sequence analysis of human mitochondrial DNA (mtDNA) is an effective and reliable tool for the genetic characterization of forensic samples. The nature of the mitochondrial genome (mtgenome), its high copy number and small size (~17kb) makes it more resistant to degradation and more stable than nuclear DNA. For this reason mitochondrial DNA is often the only feasible option for the forensic analysis of environmentally compromised samples. Currently the forensic analysis of the mtgenome is restricted to the hypervariable regions, also known as the Displacement loop (d-loop). Previous studies, confirmed in the Strausbaugh lab, have demonstrated an increased variability in the …

Metallothionein Gene Dose And The Immune Response, Meaghan Roy-O-Reilly

Honors Scholar Theses

Metallothionein (MT) is a small, cysteine-rich protein with significant immunomodulatory activity. It has been shown to play a critical role in important cellular mechanisms including heavy metal detoxification, essential metal management and the inflammatory response. MT production can be induced by a number of cellular stressors and acts to lessen the harmful effects of oxidizing agents and heavy metal exposure. Previous studies have shown that the dose of the metallothionein gene present in an individual may have significant effects on the adaptive immune response, yet the mechanism behind this phenomenon remains unknown. We hypothesize that the gene dose of metallothionein …

Identifying Progenitor Cells Of Heterotopic Ossification, Eileen E. Semancik

Honors Scholar Theses

Heterotopic Ossification (HO) is the abnormal formation of bone within extraskeletal soft tissues. The condition can occur through both genetic and acquired means. Acquired cases of HO result from invasive surgery or traumatic injuries, with increasing prevalence of ectopic skeletogenesis as a result of combat-related blast injuries. HO has been characterized to some extent, including the histological features and the mutation underlying the genetic form, but the cells resident in skeletal muscle that represent the progenitors of heterotopic bone have yet to be determined. Only a few publications have attempted to definitively determine the progenitor cells in this disorder. Findings …