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Articles 1 - 30 of 878

Full-Text Articles in Molecular Biology

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder Mar 2019

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder

Joan Lin-Cereghino

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (PAOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green ...


An Automated Bayesian Pipeline For Rapid Analysis Of Single-Molecule Binding Data, Carlas Smith, Karina Jouravleva, Maximiliaan Huisman, Samson M. Jolly, Phillip D. Zamore, David Grünwald Mar 2019

An Automated Bayesian Pipeline For Rapid Analysis Of Single-Molecule Binding Data, Carlas Smith, Karina Jouravleva, Maximiliaan Huisman, Samson M. Jolly, Phillip D. Zamore, David Grünwald

David Grünwald

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We ...


Anti-Drug Antibody Responses Impair Prophylaxis Mediated By Aav-Delivered Hiv-1 Broadly Neutralizing Antibodies, Matthew R. Gardner, Ina Fetzer, Lisa M. Kattenhorn, Meredith E. Davis-Gardner, Amber S. Zhou, Barnett Alfant, Jesse A. Weber, Hema R. Kondur, Jose M. Martinez-Navio, Sebastian P. Fuchs, Ronald C. Desrosiers, Guangping Gao, Jeffrey D. Lifson, Michael Farzan Mar 2019

Anti-Drug Antibody Responses Impair Prophylaxis Mediated By Aav-Delivered Hiv-1 Broadly Neutralizing Antibodies, Matthew R. Gardner, Ina Fetzer, Lisa M. Kattenhorn, Meredith E. Davis-Gardner, Amber S. Zhou, Barnett Alfant, Jesse A. Weber, Hema R. Kondur, Jose M. Martinez-Navio, Sebastian P. Fuchs, Ronald C. Desrosiers, Guangping Gao, Jeffrey D. Lifson, Michael Farzan

Open Access Articles

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly ...


An Automated Bayesian Pipeline For Rapid Analysis Of Single-Molecule Binding Data, Carlas Smith, Karina Jouravleva, Maximiliaan Huisman, Samson M. Jolly, Phillip D. Zamore, David Grünwald Jan 2019

An Automated Bayesian Pipeline For Rapid Analysis Of Single-Molecule Binding Data, Carlas Smith, Karina Jouravleva, Maximiliaan Huisman, Samson M. Jolly, Phillip D. Zamore, David Grünwald

Open Access Articles

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We ...


A Persistence Detector For Metabolic Network Rewiring In An Animal, Jote T. Bulcha, Gabrielle E. Giese, Zulfikar Ali, Yong-Uk Lee, Melissa D. Walker, Amy D. Holdorf, L. Safak Yilmaz, Robert C. Brewster, Albertha J. M. Walhout Jan 2019

A Persistence Detector For Metabolic Network Rewiring In An Animal, Jote T. Bulcha, Gabrielle E. Giese, Zulfikar Ali, Yong-Uk Lee, Melissa D. Walker, Amy D. Holdorf, L. Safak Yilmaz, Robert C. Brewster, Albertha J. M. Walhout

Open Access Articles

Biological systems must possess mechanisms that prevent inappropriate responses to spurious environmental inputs. Caenorhabditis elegans has two breakdown pathways for the short-chain fatty acid propionate: a canonical, vitamin B12-dependent pathway and a propionate shunt that is used when vitamin B12 levels are low. The shunt pathway is kept off when there is sufficient flux through the canonical pathway, likely to avoid generating shunt-specific toxic intermediates. Here, we discovered a transcriptional regulatory circuit that activates shunt gene expression upon propionate buildup. Nuclear hormone receptor 10 (NHR-10) and NHR-68 function together as a "persistence detector" in a type 1, coherent feed-forward loop ...


The Non-Canonical Smc Protein Smchd1 Antagonises Tad Formation And Compartmentalisation On The Inactive X Chromosome, Michal R. Gdula, Tatyana B. Nesterova, Greta Pintacuda, Jonathan Godwin, Ye Zhan, Hakan Ozadam, Michael Mcclellan, Daniella Moralli, Felix Krueger, Catherine M. Green, Wolf Reik, Skirmantas Kriaucionis, Edith Heard, Job Dekker, Neil Brockdorff Jan 2019

The Non-Canonical Smc Protein Smchd1 Antagonises Tad Formation And Compartmentalisation On The Inactive X Chromosome, Michal R. Gdula, Tatyana B. Nesterova, Greta Pintacuda, Jonathan Godwin, Ye Zhan, Hakan Ozadam, Michael Mcclellan, Daniella Moralli, Felix Krueger, Catherine M. Green, Wolf Reik, Skirmantas Kriaucionis, Edith Heard, Job Dekker, Neil Brockdorff

Open Access Articles

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells ...


Protein Degradation Regulates Phospholipid Biosynthetic Gene Expression In Saccharomyces Cerevisiae, Bryan Salas-Santiago Jan 2019

Protein Degradation Regulates Phospholipid Biosynthetic Gene Expression In Saccharomyces Cerevisiae, Bryan Salas-Santiago

Doctoral Dissertations

Transcriptional regulation of most phospholipid biosynthetic genes in Saccharomyces cerevisiae is coordinated by inositol and choline. Inositol affects phosphatidic acid (PA) intracellular levels. Opi1p interacts physically with PA and is the main repressor of the phospholipid biosynthetic genes. It is localized in the endoplasmic reticulum (ER) bound to the ER membrane protein Scs2p. When PA levels drop, Opi1p is translocated into the nucleus repressing most phospholipid biosynthetic genes. The OPI1 locus was identified in a screen looking for overproduction and excretion of inositol (Opi-). Opi- mutants are generally associated with a defect in repression of the ...


Detection Of Beta-Lactamase Variants In Municipal Wastewater And Fresh Water, Sunil Pandey Jan 2019

Detection Of Beta-Lactamase Variants In Municipal Wastewater And Fresh Water, Sunil Pandey

Masters Theses

The occurrence and spread of antibiotic-resistant genes (ARGs) are pressing public health problems worldwide. A key factor contributing to the spread of ARGs is lateral gene transfer. Wastewater treatment plants (WWTPs) are measured hot spots of microbial diversity and resistance because they receive polluted wastewater from diverse sources and contain a variety of different environments with dense bacterial loads. Due to the overuse of antibiotics the genetic capacities of microbes have profited. This helps every source of resistance gene and every means of horizontal gene transmission to develop the multiple mechanism of resistance to each antibiotic used clinically, agriculturally, or ...


Hla-Do Modulates The Diversity Of The Mhc-Ii Self-Peptidome, Padma P. Nanaware, Mollie M. Jurewicz, John D. Leszyk, Scott A. Shaffer, Lawrence J. Stern Dec 2018

Hla-Do Modulates The Diversity Of The Mhc-Ii Self-Peptidome, Padma P. Nanaware, Mollie M. Jurewicz, John D. Leszyk, Scott A. Shaffer, Lawrence J. Stern

Open Access Articles

Presentation of antigenic peptides on MHC-II molecules is essential for tolerance to self and for initiation of immune responses against foreign antigens. DO (HLA-DO in humans, H2-O in mice) is a non-classical MHC-II protein that has been implicated in control of autoimmunity and regulation of neutralizing antibody responses to viruses. These effects likely are related to a role of DO in selecting MHC-II epitopes, but previous studies examining the effect of DO on presentation of selected CD4 T cell epitopes have been contradictory. To understand how DO modulates MHC-II antigen presentation, we characterized the full spectrum of peptides presented by ...


Orbit: A New Paradigm For Genetic Engineering Of Mycobacterial Chromosomes, Kenan C. Murphy, Samantha J. Nelson, Subhalaxmi Nambi, Kadamba Papavinasasundaram, Christina E. Baer, Christopher M. Sassetti Dec 2018

Orbit: A New Paradigm For Genetic Engineering Of Mycobacterial Chromosomes, Kenan C. Murphy, Samantha J. Nelson, Subhalaxmi Nambi, Kadamba Papavinasasundaram, Christina E. Baer, Christopher M. Sassetti

Open Access Articles

Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step ...


General Decapping Activators Target Different Subsets Of Inefficiently Translated Mrnas, Feng He, Alper Celik, Chan Wu, Allan Jacobson Dec 2018

General Decapping Activators Target Different Subsets Of Inefficiently Translated Mrnas, Feng He, Alper Celik, Chan Wu, Allan Jacobson

Open Access Articles

The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C-terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and ...


Potent Cas9 Inhibition In Bacterial And Human Cells By Acriic4 And Acriic5 Anti-Crispr Proteins, Jooyoung Lee, Aamir Mir, Alireza Edraki, Bianca Garcia, Nadia Amrani, Hannah E. Lou, Ildar Gainetdinov, April Pawluk, Raed Ibraheim, Xin D. Gao, Pengpeng Liu, Alan R. Davidson, Karen L. Maxwell, Erik J. Sontheimer Dec 2018

Potent Cas9 Inhibition In Bacterial And Human Cells By Acriic4 And Acriic5 Anti-Crispr Proteins, Jooyoung Lee, Aamir Mir, Alireza Edraki, Bianca Garcia, Nadia Amrani, Hannah E. Lou, Ildar Gainetdinov, April Pawluk, Raed Ibraheim, Xin D. Gao, Pengpeng Liu, Alan R. Davidson, Karen L. Maxwell, Erik J. Sontheimer

Open Access Articles

In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K ...


Investigating Autophagy Dysfunction Induced By A Parkinson's Disease-Causing Mutation In Vps35, Abir Ashfakur Rahman Dec 2018

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 ...


Genetic And Epigenetic Investigations On Pulmonary Hypertension Syndrome In Meat Type- Chickens, Khaloud Alzahrani Dec 2018

Genetic And Epigenetic Investigations On Pulmonary Hypertension Syndrome In Meat Type- Chickens, Khaloud Alzahrani

Theses and Dissertations

This dissertation presents a collection of studies that investigate the genetic and epigenetic associations to ascites phenotype in broiler chickens. Ascites is a significant metabolic disease associated with fast-growing meat-type chickens (broilers) and is a terminal result of pulmonary hypertension syndrome PHS. It is a multi-factorial syndrome caused by interactions between genetic, physiological, environmental, and managemental factors. It was estimated that ascites accounts for losses of about US$1 billion annually worldwide and for over 25% of broilers mortality. Although traditional and molecular genetic methods in the selection and in performance improvements, has greatly reduced ascites frequency, yet it has ...


Identification Of Deubiquitinating Enzymes That Control The Cell Cycle In Saccharomyces Cerevisiae, Claudine E. Mapa Nov 2018

Identification Of Deubiquitinating Enzymes That Control The Cell Cycle In Saccharomyces Cerevisiae, Claudine E. Mapa

GSBS Dissertations and Theses

A large fraction of the proteome displays cell cycle-dependent expression, which is important for cells to accurately grow and divide. Cyclical protein expression requires protein degradation via the ubiquitin proteasome system (UPS), and several ubiquitin ligases (E3) have established roles in this regulation. Less is understood about the roles of deubiquitinating enzymes (DUB), which antagonize E3 activity. A few DUBs have been shown to interact with and deubiquitinate cell cycle-regulatory E3s and their protein substrates, suggesting DUBs play key roles in cell cycle control. However, in vitro studies and characterization of individual DUB deletion strains in yeast suggest that these ...


33 - Identification Of Proteins That Regulate Crispr Dna Uptake Of Pyrococcus Furiosus, Landon Clark Nov 2018

33 - Identification Of Proteins That Regulate Crispr Dna Uptake Of Pyrococcus Furiosus, Landon Clark

Georgia Undergraduate Research Conference (GURC)

The CRISPR-Cas (Clustered Regularly Interspace Short Palindromic Repeats-CRISPR associated) system is a prokaryotic, adaptive immune system used by bacterial and archaeal organism to fight infections by viruses and other harmful invasive DNAs. These prokaryotic CRISPR-Cas immune systems have been exploited as powerful genome editing tools that work many different organisms and cells including humans. The newly developed CRISPR-based technologies are transforming medicine and science and have been used in research applications for developing cures for certain cancers, HIV, hemophilia, etc. The function of the CRISPR-Cas systems follow three basis steps: (1) adaptation (invading DNA is integrated into the host genome ...


Rare Gene Fusion Rearrangement Sptnb1-Pdgfrb In An Atypical Myeloproliferative Neoplasm, Vanessa Fiorini Furtado, Neeraj Y. Saini, William V. Walsh, Venu G. Bathini, Patricia M. Miron Oct 2018

Rare Gene Fusion Rearrangement Sptnb1-Pdgfrb In An Atypical Myeloproliferative Neoplasm, Vanessa Fiorini Furtado, Neeraj Y. Saini, William V. Walsh, Venu G. Bathini, Patricia M. Miron

Open Access Articles

The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia recognizes a distinct class of myeloid and lymphoid tumors with eosinophilia-related proliferations associated with specific gene rearrangements, one of which involves rearrangements of platelet-derived growth factor receptor B (PDGFRB) gene. We report a case of a rare PDGFRB rearrangement with SPTNB1 (spectrin beta, nonerythrocytic 1) that presented as atypical myeloproliferative neoplasm.


Adeno-Associated Virus Neutralizing Antibodies In Large Animals And Their Impact On Brain Intraparenchymal Gene Transfer, Dan Wang, Li Zhong, Mengxin Li, Jia Li, Karen Tran, Lingzhi Ren, Ran He, Jun Xie, Richard P. Moser, Cara Fraser, Tim Kuchel, Miguel Sena-Esteves, Terence R. Flotte, Neil Aronin, Guangping Gao Oct 2018

Adeno-Associated Virus Neutralizing Antibodies In Large Animals And Their Impact On Brain Intraparenchymal Gene Transfer, Dan Wang, Li Zhong, Mengxin Li, Jia Li, Karen Tran, Lingzhi Ren, Ran He, Jun Xie, Richard P. Moser, Cara Fraser, Tim Kuchel, Miguel Sena-Esteves, Terence R. Flotte, Neil Aronin, Guangping Gao

Open Access Articles

Pre-existing neutralizing antibody (NAb) against adeno-associated virus (AAV) commonly found in primates is a major host barrier that can severely compromise in vivo gene transfer by AAV vectors. To achieve proof-of-concept success in clinical development of recombinant AAV (rAAV)-based in vivo gene therapy, it is crucial to consider the potential interference of NAb and to enroll serologically compatible study subjects. In this study, we report a large AAV NAb dataset comprising multiple large animal species and AAV serotypes and compare two NAb assays based on in vitro or in vivo transduction inhibition, respectively. Together with previously published AAV seroepidemiology ...


Characterization Of The Celf6 Rna Binding Protein: Effects On Mouse Vocal Behavior And Biochemical Function, Michael A. Rieger Aug 2018

Characterization Of The Celf6 Rna Binding Protein: Effects On Mouse Vocal Behavior And Biochemical Function, Michael A. Rieger

Arts & Sciences Electronic Theses and Dissertations

Behavior in higher eukaryotes is a complex process which integrates signals in the environment, the genetic makeup of the organism, and connectivity in the nervous system to produce extremely diverse adaptations to the phenomenon of existence. Unraveling the subcellular components that contribute to behavioral output is important for both understanding how behavior occurs in an unperturbed state, as well as understanding how behavior changes when the underlying systems that generate it are altered. Of the numerous molecular species that make up a cell, the regulation of messenger RNAs (mRNAs), the coding template of all proteins, is of key importance to ...


The Role Of Mesenchymal Stromal Cells And Classical Dendritic Cells In The Maintenance And Regulation Of The Bone Marrow Niche, Jingzhu Zhang Aug 2018

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 ...


Tumor-Stroma Interactions Differentially Alter Drug Sensitivity Based On The Origin Of Stromal Cells, Benjamin D. Landry, Thomas Leete, Ryan Richards, Peter Cruz-Gordillo, Hannah R. Schwartz, Megan E. Honeywell, Gary Ren, Alyssa D. Schwartz, Shelly R. Peyton, Michael J. Lee Aug 2018

Tumor-Stroma Interactions Differentially Alter Drug Sensitivity Based On The Origin Of Stromal Cells, Benjamin D. Landry, Thomas Leete, Ryan Richards, Peter Cruz-Gordillo, Hannah R. Schwartz, Megan E. Honeywell, Gary Ren, Alyssa D. Schwartz, Shelly R. Peyton, Michael J. Lee

Program in Systems Biology Publications and Presentations

Due to tumor heterogeneity, most believe that effective treatments should be tailored to the features of an individual tumor or tumor subclass. It is still unclear, however, what information should be considered for optimal disease stratification, and most prior work focuses on tumor genomics. Here, we focus on the tumor microenvironment. Using a large-scale coculture assay optimized to measure drug-induced cell death, we identify tumor-stroma interactions that modulate drug sensitivity. Our data show that the chemo-insensitivity typically associated with aggressive subtypes of breast cancer is not observed if these cells are grown in 2D or 3D monoculture, but is manifested ...


Microbial Ecology Of South Florida Surface Waters: Examining The Potential For Anthropogenic Influences, Chase P. Donnelly Aug 2018

Microbial Ecology Of South Florida Surface Waters: Examining The Potential For Anthropogenic Influences, Chase P. Donnelly

HCNSO Student Theses and Dissertations

South Florida contains one of the largest subtropical wetlands in the world, and yet not much is known about the microbes that live in these surface waters. These microbes play an important role in chemical cycling and maintaining good water quality for both human and ecosystem health. The hydrology of Florida’s surface waters is tightly regulated with the use of canal and levee systems run by the US Army Corps of Engineers and The South Florida Water Management District. These canals run through the Everglades, agriculture, and urban environments to control water levels in Lake Okeechobee, the Water Conservation ...


The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn Aug 2018

The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn

Electronic Theses and Dissertations

Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered through formation of complexes with close homologs. This work involves the N-terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels and in a tissue-specific manner. They are both nuclear methyltransferases with overlapping target consensus sequences ...


Deciphering The Role Of Human Arylamine N-Acetyltransferase 1 (Nat1) In Breast Cancer Cell Metabolism Using A Systems Biology Approach., Samantha Marie Carlisle Aug 2018

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 ...


Bayesian Analytical Approaches For Metabolomics : A Novel Method For Molecular Structure-Informed Metabolite Interaction Modeling, A Novel Diagnostic Model For Differentiating Myocardial Infarction Type, And Approaches For Compound Identification Given Mass Spectrometry Data., Patrick J. Trainor Aug 2018

Bayesian Analytical Approaches For Metabolomics : A Novel Method For Molecular Structure-Informed Metabolite Interaction Modeling, A Novel Diagnostic Model For Differentiating Myocardial Infarction Type, And Approaches For Compound Identification Given Mass Spectrometry Data., Patrick J. Trainor

Electronic Theses and Dissertations

Metabolomics, the study of small molecules in biological systems, has enjoyed great success in enabling researchers to examine disease-associated metabolic dysregulation and has been utilized for the discovery biomarkers of disease and phenotypic states. In spite of recent technological advances in the analytical platforms utilized in metabolomics and the proliferation of tools for the analysis of metabolomics data, significant challenges in metabolomics data analyses remain. In this dissertation, we present three of these challenges and Bayesian methodological solutions for each. In the first part we develop a new methodology to serve a basis for making higher order inferences in metabolomics ...


Computational Analysis Of Genomic Variants Affecting Predicted Microrna:Target Interactions In Prostate Cancer., Angélica Paola Hernández Pérez Jul 2018

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 ...


Production Of A Candidate Recombinant Protein Vaccine For Mannheimia Haemolytica In Lettuce And Tobacco Chloroplasts, Coby K. Martin Jul 2018

Production Of A Candidate Recombinant Protein Vaccine For Mannheimia Haemolytica In Lettuce And Tobacco Chloroplasts, Coby K. Martin

Electronic Thesis and Dissertation Repository

The cattle industry worldwide is ravaged by bovine respiratory disease (BRD), a bacterial disease caused by Mannheimia haemolytica. Recent efforts to design vaccines against M. haemolytica focus on a virulence factor, leukotoxin, in addition to surface lipoproteins. Plant-based protein production is a safe and inexpensive alternative to traditional methods. Edible vaccines deliver antigens to pharyngeal tissues, which can provide local immunization against M. haemolytica prior to its progression into the lungs. In this project, a chimeric protein containing M. haemolytica antigens was produced in tobacco chloroplasts as a candidate edible vaccine for BRD. Attempts were made to transform lettuce chloroplasts ...


Elimination Of Pcr Duplicates In Rna-Seq And Small Rna-Seq Using Unique Molecular Identifiers, Yu Fu, Pei-Hsuan Wu, Timothy J. Beane, Phillip D. Zamore, Zhiping Weng Jul 2018

Elimination Of Pcr Duplicates In Rna-Seq And Small Rna-Seq Using Unique Molecular Identifiers, Yu Fu, Pei-Hsuan Wu, Timothy J. Beane, Phillip D. Zamore, Zhiping Weng

Open Access Articles

BACKGROUND: RNA-seq and small RNA-seq are powerful, quantitative tools to study gene regulation and function. Common high-throughput sequencing methods rely on polymerase chain reaction (PCR) to expand the starting material, but not every molecule amplifies equally, causing some to be overrepresented. Unique molecular identifiers (UMIs) can be used to distinguish undesirable PCR duplicates derived from a single molecule and identical but biologically meaningful reads from different molecules.

RESULTS: We have incorporated UMIs into RNA-seq and small RNA-seq protocols and developed tools to analyze the resulting data. Our UMIs contain stretches of random nucleotides whose lengths sufficiently capture diverse molecule species ...


Effect Of Egta On Sit1 Scramblase Gene Expression And Cell Growth In Tetrahymena Thermophila, Emma Esposito, Theda Knauth, Amelia Ohnstad, Stefanie Otto-Hitt Jul 2018

Effect Of Egta On Sit1 Scramblase Gene Expression And Cell Growth In Tetrahymena Thermophila, Emma Esposito, Theda Knauth, Amelia Ohnstad, Stefanie Otto-Hitt

Life and Environmental Sciences Course-based Research Projects

Scramblase is an enzyme that facilitates the movement of newly synthesized phospholipids from the cytosolic side to the extracellular side of the lipid bilayer. This process is vital for cell membrane repair and growth. In Tetrahymena thermophila, the gene SIT1 encodes for the Scramblase protein, whose functionality is Ca2+-dependent. In this experiment, the concentration of accessible Ca2+ ions was decreased in order to observe whether the change had an aect on the expression of SIT1 and cell growth. It was hypothesized that expression of the SIT1 gene would increase, while cell growth would decrease. To carry out the experiment ...


A Rationally Engineered Capsid Variant Of Aav9 For Systemic Cns-Directed And Peripheral Tissue-Detargeted Gene Delivery In Neonates, Dan Wang, Shaoyong Li, Dominic J. Gessler, Jun Xie, Li Zhong, Jia Li, Karen Tran, Kim Van Vliet, Lingzhi Ren, Qin Su, Ran He, Jason E. Goetzmann, Terence R. Flotte, Mavis Agbandje-Mckenna, Guangping Gao Jun 2018

A Rationally Engineered Capsid Variant Of Aav9 For Systemic Cns-Directed And Peripheral Tissue-Detargeted Gene Delivery In Neonates, Dan Wang, Shaoyong Li, Dominic J. Gessler, Jun Xie, Li Zhong, Jia Li, Karen Tran, Kim Van Vliet, Lingzhi Ren, Qin Su, Ran He, Jason E. Goetzmann, Terence R. Flotte, Mavis Agbandje-Mckenna, Guangping Gao

Open Access Articles

Adeno-associated virus (AAV) has provided the gene therapy field with the most powerful in vivo gene delivery vector to realize safe, efficacious, and sustainable therapeutic gene expression. Because many clinically relevant properties of AAV-based vectors are governed by the capsid, much research effort has been devoted to the development of AAV capsids for desired features. Here, we combine AAV capsid discovery from nature and rational engineering to report an AAV9 capsid variant, designated as AAV9.HR, which retains AAV9's capability to traverse the blood-brain barrier and transduce neurons. This variant shows reduced transduction in peripheral tissues when delivered through ...