Biochemistry, Biophysics, and Structural Biology Commons^™

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 an …

Sip-428, A Sir2 Deacetylase Enzyme And Its Role In Biotic Stress Signaling Pathway, Bal Krishna Chand Thakuri

Electronic Theses and Dissertations

SABP2 (Salicylic Acid Binding Protein 2) plays a vital role in the salicylic acid signaling pathway of plants both regarding basal resistance and systemic acquired resistance against pathogen infection. SIP-428 (SABP2 Interacting Protein-428) is a Silent information regulator 2 (SIR2) like deacetylase enzyme that physically interacts with SABP2 in a yeast two-hybrid interaction and confirmed independently by a GST pull-down assay. We demonstrated that SIP- 428 is an NAD+ dependent SIR2 deacetylase enzyme. Transgenic tobacco plants silenced in SIP- 428 expression via RNAi showed enhanced basal resistance to microbial pathogens. Moreover, these SIP-428-silenced lines also exhibited a robust induction of …

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

Rowan-Virtua School of Osteopathic Medicine Departmental Research

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

Inhibition Of Ribosome Biogenesis Through Genetic And Chemical Approaches, Leonid Anikin

Graduate School of Biomedical Sciences Theses and Dissertations

In order to maintain the ability to generate proteins, proliferating cells must continuously generate ribosomes, designating up to 80% of their energy to ribosome biogenesis (RBG). RBG involves transcription of rDNA by RNA polymerases I (Pol I) and III (Pol III), expression of approximately 80 ribosomal proteins, and assembly of these components in a process referred to as ribosome maturation. During maturation, the Pol I transcribed 47S pre-rRNA undergoes a number of processing events, while simultaneously interacting with processing factors and ribosomal proteins that drive pre-ribosome assembly. Inhibition of RBG has become one of the pursued targets for cancer therapy …

Insight Into Translational Activation In Yeast Mitochondria, Julia Lynn Jones

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondrial function depends on over a thousand proteins, of which the majority are nuclear DNA-encoded and approximately one percent are mitochondrial DNA-encoded. The mitochondrial DNA of Saccharomyces cerevisiae contains eight protein-encoding genes, seven of which are required for proper function of the respiratory complexes and one encodes a ribosomal protein. The bigenomic nature of the oxidative phosphorylation complexes requires coordinated expression and regulation from both the nuclear and the mitochondrial genomes. It is currently unclear how this regulatory network operates. However, it is thought that nuclear genome-encoded messengers localized to the mitochondria aid in this coordination.

A family of proteins …

Rna-Seq Reveals Transcriptomic Program Associated With Stemness In Taxane Resistant Prostate Cancer, Christina K. Cajigas-Du Ross

Loma Linda University Electronic Theses, Dissertations & Projects

There is no cure for advanced prostate cancer (PCa), and taxane chemotherapy is the only treatment option once other therapies have failed. However, this is problematic since all patients eventually develop chemoresistance. Emerging treatments for advanced PCa have shown promise at the benchside, but clinical trials have not resulted in newly approved drugs due in part to redundant survival pathways utilized by prostate tumor cells to maintain therapy-resistance. Using RNAsequencing—an innovative approach for quantifying gene expression changes—this dissertation sought to elucidate chemoresistance-associated molecular pathways as a catalyst to develop new therapeutic targets. Results revealed a differential upregulation of stemness-associated genes …

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 …

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

Rowan-Virtua School of Osteopathic Medicine Departmental Research

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

Acetic Acid Induces Sch9p-Dependent Translocation Of Isc1p From The Endoplasmic Reticulum Into Mitochondria, António Rego, Katrina F Cooper, Justin Snider, Yusuf A Hannun, Vítor Costa, Manuela Côrte-Real, Susana R Chaves

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Changes in sphingolipid metabolism have been linked to modulation of cell fate in both yeast and mammalian cells. We previously assessed the role of sphingolipids in cell death regulation using a well characterized yeast model of acetic acid-induced regulated cell death, finding that Isc1p, inositol phosphosphingolipid phospholipase C, plays a pro-death role in this process. Indeed, isc1∆ mutants exhibited a higher resistance to acetic acid associated with reduced mitochondrial alterations. Here, we show that Isc1p is regulated by Sch9p under acetic acid stress, since both single and double mutants lacking Isc1p or/and Sch9p have the same resistant phenotype, and SCH9 …

Loss Of Function Of Gene X Protects Against Α-Dicarbonyl Stress Through The Skn-1 Pathway In C. Elegans, Austin Lim

Dissertations, Masters Theses, Capstones, and Culminating Projects

Diabetes mellitus and Parkinson’s Disease (PD) are debilitating diseases that are increasing in prevalence worldwide. One potential cause of these diseases is the accumulation of advanced glycation end products (AGEs), which are macromolecules that cause irreversible damages. AGEs are a diverse group of highly oxidative byproducts produced from α-dicarbonyl compounds (α-dcs), which are highly reactive molecules that bind indiscriminately to protein and DNA and, are regulated by a conserved glyoxalase system (GLO1 and DJ-1) in humans. Utilizing the conserved glyoxalase system, we were able to establish within a C. elegans model that when this glyoxalase system is impaired, …

Till Death Do Us Part: The Marriage Of Autophagy And Apoptosis., Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Autophagy is a widely conserved catabolic process that is necessary for maintaining cellular homeostasis under normal physiological conditions and driving the cell to switch back to this status quo under times of starvation, hypoxia, and oxidative stress. The potential similarities and differences between basal autophagy and stimulus-induced autophagy are still largely unknown. Both act by clearing aberrant or unnecessary cytoplasmic material, such as misfolded proteins, supernumerary and defective organelles. The relationship between reactive oxygen species (ROS) and autophagy is complex. Cellular ROS is predominantly derived from mitochondria. Autophagy is triggered by this event, and by clearing the defective organelles effectively, …

Functional Studies Of The E. Coli Proc And A Putative Ortholog Mrub_1345, Maureen Azar, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

This project is part of the Meiothermus ruber genome analysis project, which uses the bioinformatics tools associated with the Guiding Education through Novel Investigation –Annotation Collaboration Toolkit (GENI-ACT) to predict gene function. We investigated the biological function of Escherichia coli and Meiothermus ruber proC genes using the complementation assay. In this research project, mutants of varying severity to the functional state of the protein were developed. The results showed that two or more amino acid deletions reduced or eliminated ProC function. Amino acid substitutions, on the other hand, were not severe enough to impact ProC function. Double and triple mutants …

Determination Of Adamts13 Susceptibility In Type Iia Von Willebrand Disease, Monica Buselli

Dissertations, Masters Theses, Capstones, and Culminating Projects

von Willebrand Disease (vWD) is a bleeding disorder caused by a deficiency in von Willebrand Factor (vWF), a large glycoprotein that assists in coagulation. Specifically, large vWF multimers in the blood stream are key components in starting the coagulation cascade. vWF is cleaved by the metalloprotease ADAMTS13, regulating the multimers size, which hinders vWF’s ability to function properly. The three main types of vWD —Type I, II, and III— are not well defined, and as a result are all similarly treated with plasma-derived vWF replacement therapy. Plasma-derived vWF is a treatment that does not cure the problem but relieves the …

The Characterization Of The Transcription Factor Msab And Its Role In Staphylococcal Virulence, Justin Batte

Dissertations

Staphylococcus aureus is a common human pathogen that is responsible for a wide range of infections, ranging from relative minor skin infections to life-threatening disease such as bacteremia, septicemia, and endocarditis. S. aureus possesses many different virulent factors that aid in its ability to cause this wide array of infections. One major virulence factor includes the production of capsular polysaccharide (CP). The production of CP plays a major role in the virulence response during infection specifically by providing S. aureus an antiphagocytic mechanism that allows the pathogen to evade phagocytosis during an infection. S. aureus has developed complex genetic regulatory …

Functional And Structural Impact Of The Loss Of The Leucine-Rich Repeat Protein Lrit1 In The Mouse Retina., Catherine Ann Cobb

Electronic Theses and Dissertations

Mutations in genes encoding the leucine-rich repeat (LRR) proteins nyctalopin and LRIT3 lead to complete congenital stationary night blindness because they are critical to depolarizing bipolar cell function in the retina. LRIT3 has two closely related family members, LRIT1 and LRIT2. In silico analyses of publicly available RNA-Seq data showed that Lrit1 was highly expressed in the retina. Here I describe the expression pattern and impact of loss of LRIT1 on retinal function. To enable these studies, we used CRISPR/Cas9 technology to create an Lrit1^-/- mouse line. Retinal morphology and morphometry analyses showed no gross changes in retinal structure …

Regulation Of The Tubulin Homolog Ftsz In Escherichia Coli, Monika S. Buczek

Dissertations, Theses, and Capstone Projects

Escherichia coli is a well-known pathogen, and importantly, a widely used model organism in all fields of biological sciences for cloning, protein purification, and as a model for Gram-negative bacterial species. And yet, researchers do not fully understand how this bacterium replicates and divides. Every year additional division proteins are discovered, which adds complexity to how we understand E. coli undergoes cell division. Due to their specific roles in cytokinesis, some of these proteins may be potential targets for development of antibacterials or bacteriostatics, which are much needed for fighting the current global antibacterial deficit. My thesis work focuses on …

Involvement Of The Ino80 Complex In Chromosome Segregation, Jesus Moreno

Graduate Theses and Dissertations

Chromatin remodeling complexes are multi-protein complexes that regulate the dynamics of the nucleosomes in the genome. The INO80 chromatin remodeling complex participates in varied biological processes such as: transcription, DNA repair, DNA replication and chromosome integrity. It catalyzes the eviction of the H2A.Z variant histone as well as whole nucleosome eviction. This complex is comprised of 15 subunits and the contribution of each to chromosome segregations remains unknown. To evaluate the contribution of each subunit to chromosome segregation, we tested deletion mutants of the non-essential subunits for DNA content and benomyl sensitivity. Also, we assessed members of the SWR1 and …

Development Of Endogenous Tagging Plasmids For Characterization Of Protein Interactions, Localization, And Post-Translational Modifications Of Tetrahymena Thermophila Rad23, Evan Andrew Wilson

Graduate Theses/Dissertations

Rad23 is a protein involved in both nucleotide excision repair (NER) and proteasome-mediated degradation, and has been suggested to facilitate interactions between these two pathways. The model organism Tetrahymena thermophila, which has a transcriptionally silent micronucleus, provides a useful platform for studying the role of Rad23 in global genome NER (GG-NER). However, the ectopic expression systems used thus far in T. thermophila to study Rad23 are repressed by UV light and do not account for the background expression of endogenous RAD23; these phenomena prevent insightful gains to the true dynamics of Rad23. In this thesis, endogenous tagging …

Evolution Via Gene Duplication And Alternative Splicing In The Eukaryotic Ski7 And Hbs1 Genes, Alexandra Marshall

Dissertations and Theses (Open Access)

Gene duplication and alternative splicing are both recognized as important drivers of proteomic diversity and innovation during evolution, but the evolutionary changes over long periods of time or the interrelations of the two processes has not been extensively studied. Here I study these phenomena for the SKI7 and HBS1 gene pair. These Saccharomyces cerevisiae genes were created as part of a whole genome duplication (WGD) event and have since functionally diverged. Although both genes function in mRNA surveillance pathways, the two genes act on different RNAs and have different effects on the target mRNAs. Ski7 brings the Ski complex and …

Functional Similarity Of Prd-Containing Virulence Regulators In Bacillus Anthracis, Malik Raynor

Dissertations and Theses (Open Access)

Bacillus anthracis produces three regulators, AtxA, AcpA, and AcpB, that control virulence gene expression and are members of an emerging class of regulators termed “PCVRs” (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA controls expression of the toxin genes; lef, cya, and pag, and is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA activity result in capBCADE transcription in strains lacking acpA and acpB. RNA-Seq was used to assess the regulons of the …

Investigating The Impact Of Intragenic Dna Methylation On Gene Expression, And The Clinical Implications On Tumor Cells And Associated Stroma, Michael Mcguire

Dissertations and Theses (Open Access)

Investigations into the function of non-promoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated non-promoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we uncovered an enrichment of DNA methylation sites within the gene body and 3’UTR in which DNA methylation is strongly positively correlated with gene expression. We examined 32 tumor types and identified 57 …

The Regulation Of Dna Methylation In Mammalian Development And Cancer, Nicolas Veland

Dissertations and Theses (Open Access)

DNA methylation is an essential epigenetic modification in mammals, as it plays important regulatory roles in multiple biological processes, such as gene transcription, maintenance of chromosomal structure and genomic stability, genomic imprinting, retrotransposon silencing, and X-chromosome inactivation. Dysregulation of DNA methylation is associated with various human diseases. For example, cancer cells usually show global hypomethylation and regional hypermenthylation, which have been implicated in genomic instability and tumor suppressor silencing, respectively. Although great progress has been made in elucidating the biological functions of DNA methylation over the last several decades, how DNA methylation patterns and levels are regulated and dysregulated is …

Unravelling The Layers Of Cell Wall Synthesis And Function In Rice, Ritu Mihani

Graduate Theses and Dissertations

The plant cell wall is of critical importance to plant growth and survival, functioning in maintaining structural integrity, supporting cell expansion, and acting as the first line of defense in response to biotic and abiotic stresses. The major components of the cell wall are cellulose, hemicelluloses, lignin, and pectin. Recent focus on the transcriptional machinery regulating cell wall biosynthesis in plants has revealed many key transcription factors responsible for orchestrating cell wall deposition. However, many of these TFs act redundantly and work coherently with a suite of TFs to activate the cell wall biosynthetic machinery. Heterologous expression of TFs is …

Effect Of Mutation Of Cvn7 Conservon Genes Sco6796 And Sco6798 Of Streptomyces Coelicolor., Caroline Fulmer

Undergraduate Distinction Papers

Abstract

Streptomyces coelicolor is a gram-positive Actinobacterium. It is a filamentous soil organism that performs complex multicellular development. Its physiological and morphological differentiation produces active secondary metabolite antibiotics that have pharmaceutical applications. Studies of S. coelicolor show that cyclic di-GMP proteins regulate physiological and morphological development. S. coelicolor has 13 copies of an operon that are collectively named “conservon” because they are highly conserved. The operons are named cvn1-13 and genes in each operon are denoted cvnA-D. RNA-Seq data of a cyclic di-GMP phosphodiesterase double mutant showed differential expression between wild type and many of the operons in the …

Identifying Rmda Protein Interactions In Streptomyces Using A Bacterial Two-Hybrid System, Rachel Nguyen

Undergraduate Distinction Papers

Streptomyces is a genus of the phylum actinobacteria most commonly found as soil bacteria and used as a major source of antibiotics. RmdA and RmdB are phosphodiesterases that break down the ubiquitous second messenger cyclic-di-GMP which determines colony morphology and development of Streptomyces. The objective of this research is to identify whether RmdA will have interactions with itself using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) System. Each gene was fused into one of two BACTH vectors that encode a different domain of a single protein (T18 and T25) and then cotransformed into the BACTH indicator strain. The transformants were …

The Identification And Characterization Of A Putative Chromosome Segregation Gene In Streptomyces Coelicolor, Sean Kirk

Undergraduate Distinction Papers

Streptomyces coelicolor is a soil bacterium that is a model for bacterial development. It is a filamentous, sporulating bacterium known to produce many medically utilized antibiotics. The goal of this research was to examine several developmental mutants and characterize novel genes of interest. Previously generated random transposon insertion mutants were analyzed using visual and microscopic phenotyping. Mutants of interest were further pursued and each transposon disruption site was identified by Inverse PCR and DNA sequencing. One of the novel genes is suspected to be involved in DNA segregation and codes for a putative membrane protein. Staining with propidium iodide was …

Copy Number Variation In The Porcine Genome Detected From Whole-Genome Sequence, Rebecca Anderson

Honors Program: Senior Projects (Public)

Copy number variations (CNVs) are large insertions, deletions, and duplications in the genome that vary between individuals in a species. These variations are known to impact a broad range of phenotypes from molecular-level traits to higher-order clinical phenotypes. CNVs have been linked to complex traits in humans such as autism, attention deficit hyperactivity disorder, nervous system disorders, and early-onset extreme obesity. In this study, whole-genome sequence was obtained from 72 founders of an intensely phenotyped experimental swine herd at the U.S. Meat Animal Research Center (USMARC) in Clay Center, Nebraska. This included 24 boars (12 Duroc and 12 Landrace) and …

Examination Of Orthologous Genes (Mrub_2518 And B3728, Mrub_2519 And B3727, Mrub_2520 And B3726, Mrub_2521 And B3725) Responsible For Abc Phosphate Transporters In Two Species M. Ruber And E. Coli, Margaret Meyer, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

In this project we investigated the biological function of the genes b3725, b3726, b3727, b3728 and Mrub_2518, Mrub_2519, Mrub_2520 and Mrub_2521 (KEGG map number 02010). We predict that these genes encode the components of a Phosphate ABC transporter: Orthologous genes Mrub_2518 (DNA coordinates 2565359..2566438) and b3728 encodes the periplasmic phosphate binding component; Orthologous genes Mrub_2519 (DNA coordinates 2566499..2567485) and b3727, and Mrub_2520 (DNA coordinates 2567496..2568326) and b3726 encode for the two transmembrane proteins; Orthologous genes Mrub_2521 (DNA coordinates 2568338..2569159) and b3725 encode for the ATP binding protein within the cytoplasm. Within the two species, M. ruber and E. coli, …

Mrub_1325, Mrub_1326, Mrub_1327, And Mrub_1328 Are Orthologs Of B_3454, B_3455, B_3457, B_3458, Respectively Found In Escherichia Coli Coding For A Branched Chain Amino Acid Atp Binding Cassette (Abc) Transporter System, Bennett Tomlin, Adam Buric, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

In this project we investigated the biological function of the genes Mrub_1325, Mrub_1326, Mrub_1327, and Mrub_1328 (KEGG map number 02010). We predict these genes encode components of a Branched Chain Amino Acid ATP Binding Cassette (ABC) transporter: 1) Mrub_1325 (DNA coordinates 1357399-1358130 on the reverse strand) encodes the ATP binding domain; 2) Mrub_1326 (DNA coordinates 1358127-1359899 on the reverse strand) encodes the ATP-binding domain and permease domain; 3) Mrub_1327 (DNA coordinates 1359899-1360930 on the reverse strand) encodes a permease domain; and 4)Mrub_1328 (DNA coordinates 1711022-1712185 on the reverse strand) encodes the substrate binding domain. This system is not predicted to …

Confirmation That Mrub_1751 Is Homologous To E. Coli Xylf, Mrub_1752 Is Homologous To E. Coli Xylh, And Mrub_1753 Is Homologous To E. Coli Xylg, Ben Price, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

In this project we investigated the biological function of the genes Mrub_1751, Mrub_1752 and Mrub_1753 (KEGG map number 02010). We predict these genes encode components of a D-xylose ATP Binding Cassette (ABC) transporter: 1) Mrub_1752 (DNA coordinates 1809004-1810224 on the forward strand) encodes the permease component (aka transmembrane domain), predicted to be an ortholog and 2) Mrub_1753 (DNA coordinates 1810227-1811000 on the forward strand) encodes the ATP-binding domain (aka nucleotide binding domain); and 3) Mrub_1751 (DNA coordinates 1807855-1808892 on the forward strand) encodes the solute binding protein. The ABC-transporter for M. ruber to transport D-xylose is homologous with the transporter …