Biochemistry Commons^™

Understanding Ten-Eleven Translocation-2 In Hematological And Nervous Systems, Feng Pan

FIU Electronic Theses and Dissertations

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems.

In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA …

P120-Catenin Regulates Rest And Corest, And Modulates Mouse Embryonic Stem Cell Differentiation, Moonsup Lee

Dissertations & Theses (Open Access)

The canonical-Wnt pathway and beta-catenin have been extensively studied to determine their contributions to stem cell biology, but less is known about p120-catenin in the nuclear compartment. P120 is developmentally required as a consequence of its biochemical and functional interactions with cadherins, small-GTPases and transcriptional regulators. We report here that p120-catenin binds to and negatively regulates REST and CoREST, that others have indicated form a repressive complex having diverse key roles in developmental and pathologic gene regulation. We thus provide the first evidence for a direct upstream modulator of REST/CoREST function. Using mouse embryonic stem cells (mESCs), mammalian cell lines, …

Nuclear Transport Of Single Molecules: Dwell Times At The Nuclear Pore Complex, Ulrich Kubitscheck, David Grunwald, Andreas Hoekstra, Daniel Rohleder, Thorsten Kues, Jan Peter Siebrasse, Reiner Peters

David Grünwald

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of …

Intranuclear Binding Kinetics And Mobility Of Single Native U1 Snrnp Particles In Living Cells, David Grunwald, Beatrice Spottke, Volker Buschmann, Ulrich Kubitscheck

David Grünwald

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay …

Autonomy And Robustness Of Translocation Through The Nuclear Pore Complex: A Single-Molecule Study, Thomas Dange, David Grunwald, Antje Grunwald, Reiner Peters, Ulrich Kubitscheck

David Grünwald

All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapalpha2, kapbeta1, kapbeta1DeltaN44, and kapbeta2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy …

Nuclear Pore Component Nup98 Is A Potential Tumor Suppressor And Regulates Posttranscriptional Expression Of Select P53 Target Genes, Stephan Singer, Ruiying Zhao, Anthony M. Barsotti, Anette Ouwehand, Mina Fazollahi, Elias Coutavas, Kai Breuhahn, Olaf Neumann, Thomas Longerich, Tobias Pusterla, Maureen A. Powers, Keith M. Giles, Peter J. Leedman, Jochen Hess, David Grunwald, Harmen J. Bussemaker, Robert H. Singer, Peter Schirmacher, Carol Prives

David Grünwald

The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3'UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3sigma) to be similarly regulated by Nup98. The expression …

Poly(Arginine) Derived Cancer-Targeting Peptides For The Development Of A Cancer-Targeted Gene Therapy Approach In Hepg2 Liver Cancer Cells, Stesha C. Joseph

Seton Hall University Dissertations and Theses (ETDs)

Cancer is a disease that has eluded medicinal approaches for many years and as a result new and improved therapeutic approaches are in constant demand. Although chemotherapy and radiation treatments have assisted in suppressing the growth of tumors, their poor selectivity and efficacy are major limitations for effective therapy en route towards the development of a cure for the cancer epidemic. With the mission of conquering cancer at heart, researchers have pursued a new form of cancer therapy, aptly named, a cancer targeting approach. This method revolves around the selection of a suitable biomarker, typically a cell surface receptor …

Mapping The Human Vasculature By In Vivo Phage Display, Julianna Bronk

Dissertations & Theses (Open Access)

In vivo phage display screenings by intravenous injection of a random phage-displayed peptide library allow for the selection of peptides that localize to specific vascular beds. At the University of Texas MD Anderson Cancer Center, we have had the opportunity to perform phage display screenings in cancer patients in order to select for cancer specific targets directly in humans. These targets serve to define biochemical diversity of endothelial cell surfaces and can be validated and explored towards the design of vascular-targeted pharmacology. In the most recent patient screen, samples were recovered from hepatocellular carcinoma (HCC) as well as 26 additional …

Angiomotin Is A Novel Cadherin-11 Interacting Protein That Mediates Migration In Prostate Cancer Cells, Angelica Ortiz

Dissertations & Theses (Open Access)

Prostate cancer (PCa), the second leading cause of cancer-related deaths among men in the United States, has the proclivity to metastasize to bone resulting in sclerotic lesions. These cancer induced bone growths cause bone pain and fractures. Therefore, understanding the molecular mechanisms contributing to PCa bone metastasis is required in order to find better prognostic tools and suitable targets for metastasis treatment and/ or prevention. Previous work in our laboratory showed increased expression of cadherin-11 (Cad11), a mesenchymal cadherin, during PCa progression. Furthermore, Cad11 expression endows PCa cells with increased migratory potential and metastasis to bone. Deletion of the Cad11 …

Experimental Demonstration Of Bindingless Signal Delivery In Human Cells Via Microfluidics, Fang-Tzu Chuang

Fang-Tzu Chuang

The cellular signal transduction is commonly believed to rely on the direct “contact” or “binding” of the participating molecule reaction that depends positively on the corresponding molecule concentrations. In living systems, however, it is somewhat difficult to precisely match the corresponding rapid “binding,” depending on the probability of molecular collision, existing in the cellular receptor-ligand interactions. Thus, a question arises that if there is another mechanism (i.e., bindingless) that could promote this signal communication. According to this hypothesis, we report a cellular model based on the examination of intracellular calcium concentration to explore whether the unidentified signal delivery in cells …

Key Residues Of Human Cytoplasmic Protein Tyrosine Phosphatase-A And -B For Substrate Binding And Specificity, Byunghyun Park

Open Access Theses

Reversible tyrosine phosphorylation plays an important role in signaling pathways that are essential for regulating cellular growth, differentiation and metabolism. Moreover, several human diseases such as diabetes, obesity and cancers are associated with the deregulation of protein tyrosine phosphatases (PTPs). Several studies provide evidence that PTPs not only contribute to cellular differentiation, but over-expression of these molecules also leads to transformation of non-transfomed cells as well. Based on these results, designing specific PTP inhibitors may ultimately function as potential therapeutic agents to treat various diseases including cancer, diabetes, and autoimmune diseases. EphA2 is a receptor tyrosine kinase which is hypo-phosphorylated …

Experimental And Computational Analysis Of The Synucleins, Agatha Munyanyi

Theses and Dissertations in Biomedical Sciences

The synuclein proteins α, β and γ which are located in the brain, have been a subject of intense research. Of particular interest is α-synuclein, which is found in misfolded forms in Lewy bodies that are associated with Parkinson's disease. Despite the efforts of researchers across the world, the physiological structure and function of the synucleins remains elusive. In recent years, highly controversial reports by some investigators indicate that in its natural form, α-synuclein exists as a tetramer instead of as an intrinsically unstructured monomer. This dissertation presents results of the experimental and computational analysis of the synucleins. First, we …

Branching Into Rnai: Synthesis, Characterization And Biology Of Branch And Hyperbranch Sirnas, Anthony Muriithi Maina

Seton Hall University Dissertations and Theses (ETDs)

The cancer epidemic continues to afflict millions of humans world-wide each year and despite a renewed hope with the development of new and improved forms of therapy, a cure for cancer remains an elusive goal. This is partly related to the rise of resilient forms of tumors that have evolved with resistance towards conventional chemotherapy and radiation treatments. Moreover, these non-specific therapeutic regimens are highly toxic, leading to severe immunosuppressive effects which poisons the body and compromises the road towards remission. In an effort to mitigate these limitations, cancer-targeting approaches are currently experiencing a renaissance in the translation of new …

Diabetes And Obesity Induce Transcriptomic And Metabolomic Changes Enhancing Pancreatic Cancer Aggressiveness, Guermarie Velázquez Torres

Dissertations & Theses (Open Access)

Pancreatic cancer is one of the most aggressive types of cancer, with poor prognosis that lacks effective diagnostic markers and therapies. It is expected that in 2014 the incidence and the mortality of pancreatic cancer in the United States will be 46,420 and 39,590 respectively. Diabetes and obesity are modifiable risk factors associated with accelerated pancreatic carcinogenesis and tumor progression, but the biological mechanisms are not completely understood. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating these epidemiologic phenomena. Our hypothesis is that obesity and diabetes mellitus type 2 (DM2) accelerate pancreatic cancer and …

Phthalates And Phthalate Alternatives: Effects On Proliferative And Estrogenic Target Genes In Ishikawa Cells, Ranjani Sundar '15, Ping Yin, Serdar E. Bulun

Student Publications & Research

Phthalates are used as plasticizers in many of the products found in medical, household, and industrial applications. Much research has not been completed on the effects of these phthalates as potential endocrine disrupting chemicals (EDCs). As these chemicals are ingested, the mechanism by which they affect the reproductive system is largely unknown. The purpose of this study was to observe how 2 phthalates, Di-n-butyl phthalate (DBP) and Diisononyl phthalate (DINP), and 2 phthalate alternatives, Dioctyl terephthalate (DOTP) and BHT (butylated hydroxytoluene)affect uterine cells in comparison to a vehicle treatment and 17β-Estradiol treatment. Changes in expression of mRNA were observed using …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

University Scholar Projects

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Defining The Sites Of Interaction Of The Fancd2, Fance, And Fancl Proteins, Joseph Mcclanaghan

Senior Honors Projects

Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure and increased cancer susceptibility. FA is caused by mutations in any one of 16 genes. These genes encode for proteins that function in the FA-BRCA pathway to repair damaged DNA. Because of its important r­­­ole in DNA repair, this pathway is considered a major cellular tumor suppressor pathway, i.e. is critical for the prevention of cancer. Underscoring this fact, several of the FA genes - including BRCA2, BRIP1, PALB2, and RAD51C - are bona fide breast and ovarian cancer susceptibility genes.

My …

Functional Analysis Of Cytosolic Hsp70 Nucleotide Exchange Factor Networks In Yeast, Jennifer Lynn Abrams

Dissertations & Theses (Open Access)

The Hsp70 class of molecular chaperones play critical roles in protein homeostasis via an ATP-dependent folding cycle. Cytosolic Hsp70s in the budding yeast Saccharomyces cerevisiae, Ssa and Ssb, interact with up to three distinct nucleotide exchange factors (NEFs) homologous to human counterparts; Sse1/Sse2/HSP110, Fes1/HspBP1, and Snl1/Bag1. In an effort to understand the differential functional contributions of the cytosolic NEFs to protein homeostasis (“proteostasis”), I carried out comparative genetic, biochemical and cell biological analyses. For these studies, I developed protocols to monitor protein disaggregation and reactivation in a near real-time coupled assay that revealed the importance of aggregate dynamics in the …

Examining The Functional Consequences Of The Flexibility Of Aminoglycoside Phosphotransferase (3’)-Iiia, Katelyn Dawn Rosendall

Masters Theses

The use of aminoglycoside antibiotics began in 1940 with the discovery of streptomycin. The overuse and misuse of antibiotics has resulted in prevalent cases of antibiotic resistance. The most common source of aminoglycoside resistance is the presence of enzymes that covalently modify the antibiotics at specific locations. One such enzyme, APH(3′)-IIIa [the aminoglycoside phosphotransferase three prime three a] conveys resistance by transferring the γ-phosphate [gamma phosphate] from ATP [adenosine triphosphate] onto the 3′ [three prime] carbon of the aminoglycoside antibiotic sugar ring. APH(3′)-IIIa has been shown to be flexible in solution and this flexibility is proposed to be responsible for …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Molecular Chaperone Tools For Use Against Neurodegenerative Diseases, Matthew Tinkham

Senior Honors Projects

A noted characteristic found in several neurodegenerative disorders, including Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease and bovine spongiform encephalopathy, is the accumulation of amyloid plaques in the brain. Amyloid plaques contain deposits of fibrillar aggregates of misfolded proteins that disrupt normal functionality in neurons. Certain variants of these misfolded proteins are self-replicating; these self-replicating amyloids are termed prions (for infectious protein). We are interested in how protein misfolding contributes to amyloid formation and how molecular chaperone proteins can change the formation of amyloid deposits. Chaperone proteins function by catalyzing the proper folding of other proteins, the refolding of misfolded proteins, …

Characterization Of Ftsa-Ftsn Interaction During Escherichia Coli Cell Division, Kimberly.Busiek@Gmail.Com K. Busiek

Dissertations & Theses (Open Access)

Division of a bacterial cell into two equal daughter cells requires precise assembly and constriction of the division machinery, or divisome. The Escherichia coli divisome includes nearly a dozen essential cell division proteins that assemble at midcell between segregating sister chromosomes. FtsZ, a homolog of eukaryotic tubulin, is the first essential cell division protein to localize at midcell where it polymerizes into a ring-shaped scaffold (Z ring). Establishment of the Z ring is required for recruitment of downstream cell division proteins including FtsA, a cytoplasmic protein that tethers the Z ring to the inner membrane. Following localization of FtsA and …

Maternal Control Of Genomic Imprinting: Effects Of Infertility And Ovarian Stimulation In A Mouse Model, Michelle M. Denomme

Electronic Thesis and Dissertation Repository

Gametogenesis and early embryogenesis are important stages in which genome-wide epigenetic transitions required for early mammalian development are orchestrated. This is exemplified by the occurrence of genomic imprinting, where epigenetic mechanisms lead to the monoallelic expression of a subset of genes. Parental-specific DNA methylation in the gametes results in the distinct nonequivalence of the parental genomes in the early embryo. Changes from normal gamete and embryo development by impaired fertility or assisted reproductive technologies (ARTs) may disrupt the processes of imprint acquisition and imprint maintenance. My hypothesis is that aberrant imprinted methylation arises from impaired maternal fertility or ovarian stimulation …

Draft Genome Sequence Of A Stable Mucoid Strain Of Pseudomonas Aeruginosa Pao581 With A Muca25 Mutation, Yeshi Yin, T. Ryan Withers, John R. W. Govan, Shannon L. Johnson, Hongwei D. Yu

Hongwei Yu

A mutation in the mucA gene, which encodes a negative regulator of alginate production in Pseudomonas aeruginosa, is the main mechanism underlying the conversion to mucoidy in clinical isolates from patients with cystic fibrosis (CF). Here, we announce the draft genome sequence of the stable alginate-overproducing mucoid strain P. aeruginosa PAO581 with a mucA25 mutation, a derivative from the nonmucoid strains P. aeruginosa PAO381 and PAO1.

Exploration Of Mutations In Erythroid 5-Aminolevulinate Synthase That Lead To Increased Porphyrin Synthesis, Erica Jean Fratz

USF Tampa Graduate Theses and Dissertations

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first committed step of heme biosynthesis in animals, the condensation of glycine and succinyl-CoA yielding 5-aminolevuliante (ALA), CoA, and CO2. Murine erythroid-specific ALAS (mALAS2) variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. Transfection of HeLa cells with expression plasmids for mALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatment of HeLa cells expressing mALAS2 variants revealed …

Metabolic Checkpoints In Cancer Cell Cycle, Mahesh Saqcena

Dissertations, Theses, and Capstone Projects

Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from …

Lipid Dependence In Ras-Driven Tumors, Darin Salloum

Dissertations, Theses, and Capstone Projects

Over past decade, metabolic alterations in cancer cells have received a substantial amount of interest. It had been established that cancer cells undergo a significant amount of metabolic alterations, and some of these alterations are similar to those in normal highly proliferative cells. However, it is becoming more apparent that many of the metabolic alterations are specific to particular oncogenic signaling pathways. Although altered metabolic machinery makes cancer cells more efficient at promoting growth when nutrients are supplied at the sufficient amounts, the dependency of cancer cells on particular metabolic reprogramming deems cancer cells susceptible to disruptions within metabolic network. …

Identification And Characterization Of Protein Phopshatases Regulating The Sma/Mab Pathway In C. Elegans, Sheng Xiong

Dissertations, Theses, and Capstone Projects

TGF-beta signaling is a conserved signaling pathway among eukaryotes, which controls various normal cellular responses from cell proliferation to cell death. The mutations in its components are found in developmental disorders and cancer. Therefore, this signaling pathway is extensively investigated so that new therapeutic targets could be discovered and novel drugs could be developed. Previous studies suggested the involvement of phosphatases in regulation of TGF-beta signaling, but these studies were performed in cell culture rather than intact organisms. C. elegans is a tractable organism in which to study signaling in vivo. In C. elegans, growth is controled by a conserved …

Effect Of A 10 Day Decrease In Physical Activity On Circulating Angiogenic Cells, Gayatri Guhanarayan

Masters Theses 1911 - February 2014

Circulating angiogenic cells (CACs) are early predictors of cardiovascular health and are inversely proportional to related outcomes. Increased number and function of CACs is seen in healthy individuals compared with individuals with cardiovascular disease (CVD). Exercise increases CAC number and function in CVD populations, through a nitric oxide-mediated mechanism. Inactivity is a growing concern in industrialized nations; it is an independent risk factor for CVD and is linked to increased mortality. The purpose of this study was to understand the effect of reduced physical activity (rPA) on two CAC populations (CFU-Hill and CD34⁺) in highly active individuals. We …

Cellular Regulation Of Extension And Retraction Of Pseudopod-Like Blebs Produced By Nanosecond Pulsed Electric Field, Mikhail A. Rassokhin, Andrei G. Pakhomov

Bioelectrics Publications

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca²⁺ -free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca²⁺ , cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after …