Biochemistry Commons^™

Characterization Of Lignin Structural Variability And The Associated Application In Genome Wide Association Studies, Nathan D. Bryant

Doctoral Dissertations

Poplar (Populus sp.) is a promising biofuel feedstock due to advantageous features such as fast growth, the ability to grow on marginal land, and relatively low lignin content. However, there is tremendous variability associated with the composition of biomass. Understanding this variability, especially in lignin, is crucial to developing and implementing financially viable, integrated biorefineries. Although lignin is typically described as being comprised of three primary monolignols (syringyl, guaiacyl, p-hydroxyphenyl), it is a highly irregular biopolymer that can incorporate non-canonical monolignols. It is also connected by a variety of interunit linkages, adding to its complexity. Secondary cell wall …

Upland Cotton And Nematodes: An Analysis Of Historical Resistance, Upcoming Threats, And Co-Inoculation Effects, Amanda Gaudin

Theses and Dissertations

Upland cotton (Gossypium hirsutum ) is an important fiber crop grown throughout the southern United States. Plant-pathogenic nematodes are worm-like animals that feed on the roots of most agronomic crops, including cotton. The southern root-knot nematode (Meloidogyne incognita, RKN) and the reniform nematode (Rotylenchulus reniformis, RN) cause significant yield losses in cotton every year. Current sources of resistance are effective but limited, therefore historical screenings of cotton accessions were revisited in search for novel resistance sources. None were identified but many of the screened accessions possessed markers of known root-knot nematode and reniform nematode resistance. …

Advances In Phaeodactylum Tricornutum Nuclear Engineering, Mark Pampuch

Electronic Thesis and Dissertation Repository

The marine diatom Phaeodactylum tricornutum has the potential to become an excellent platform for the sustainable production of valuable compounds and pharmaceuticals, but currently large-scale engineering of this organism remains a challenge due factors like inefficient genetic transformation protocols and a lack of accurate genomic data. This thesis addresses these two bottlenecks by (i) optimizing an electroporation protocol to P. tricornutum and (ii) remapping genomic data from a scaffolded genome assembly to a telomere-to-telomere genome assembly. An optimized transformation protocol was developed that could consistently transform blunt-ended and DNA with overhangs and yielded up to 1000+ colony forming units per …

Acetate Metabolism In The Fungal Pathogen Cryptococcus Neoformans, Oly Ahmed

All Dissertations

Cryptococcus neoformans is an environmental basidiomycetous fungus with a worldwide distribution and a wide range of habitats. Inhalation of the desiccated yeasts or spores of C. neoformans often leads to opportunistic pulmonary infections in immunocompromised individuals, and in severe cases causes lethal meningitis following hematogenous dissemination. During infection, depending on the tissue and disease state, the invading fungi experience a range of nutrient microenvironments within the host body. As a result, rapid metabolic adaptations geared towards efficient utilization of carbon sources alternative to glucose become one of the prime determinants of survival and growth for the pathogen. Incidentally, cryptococcal infection …

Towards More Complete Metagenomic Analyses Through Circularized Genomes And Conjugative Elements, Benjamin R. Joris

Electronic Thesis and Dissertation Repository

Advancements in sequencing technologies have revolutionized biological sciences and led to the emergence of a number of fields of research. One such field of research is metagenomics, which is the study of the genomic content of complex communities of bacteria. The goal of this thesis was to contribute computational methodology that can maximize the data generated in these studies and to apply these protocols human and environmental metagenomic samples.

Standard metagenomic analyses include a step for binning of assembled contigs, which has previously been shown to exclude mobile genetic elements, and I demonstrated that this phenomenon extends to all conjugative …

Investigating The Biochemical Properties Of A Novel Mutation, A194v, In Human Rad51, Briana Vollbeer

All Theses

DNA double-strand breaks (DSB) are one of the most serious DNA lesions because improper repair of a DSB can lead to loss of heterozygosity, aneuploidy, and cancer. One of the primary pathways to repair DSBs is homologous recombination (HR). HR resects the DNA around the DSB and then uses homologous DNA as a template to restore the broken sequence. RAD51 has a vital function in this pathway by forming a nucleoprotein filament on a resected end of the DSB. The nucleoprotein filament searches for homology within the homologous DNA. Once homology is located, strand invasion followed by strand exchange occurs. …

Investigating The Biorisk Of Genetically Engineered Thermosynechococcus Elongatus Bp1, Cherrelle Leah Barnes

Chemistry & Biochemistry Theses & Dissertations

Cyanobacteria, also known as blue-green algae, are an ancient group of microorganisms that use simple materials, such as sunlight, carbon dioxide and water, to produce energy while providing oxygen to the atmosphere by performing photosynthesis. Synthetic biology approaches have been employed with cyanobacteria as a platform to produce a range of products, such as biofuels, by inserting a series of genes into the cyanobacterial genome that will allow the conversion of metabolic intermediates to such desired products. Although these methods are promising, it is important to understand any potential bio-risk they pose. This research evaluates the potential bio-risk of genetically …

Mechanisms Of Telomere Maintenance In Trypanosoma Brucei, M A G G. Rabbani

ETD Archive

Telomeres are a nucleoprotein structure at the end of the chromosome and are essential for genome integrity and chromosome stability. Telomere lengths are primarily maintained by a telomerase-mediated pathway but can be maintained by a homologous recombination-mediated pathway. However, detailed mechanisms of telomere maintenance are still unclear in many eukaryotes, including an important human pathogen, Trypanosoma brucei. Telomeres can be elongated by telomerase in T. brucei, a causative agent of fatal sleeping sickness in humans and nagana in cattle. T. brucei evades host immune response by regularly switching its major surface antigen, variant surface glycoprotein (VSG), a process known as …

Characterization Of A Potential Glucose Transporter In Trypanosoma Brucei, Matthew Morgan

All Theses

Trypanosoma brucei, the African trypanosome, is an organism heavily dependent on glucose for ATP production during the infectious stage of its life cycle. Here, we have explored the role of an uncharacterized protein designated “novel glucose transporter” (NGT) as a potential glucose transporter. Sequence analyses suggests that NGT shares similarities (either at the primary sequence level or structurally) with Trypanosome Hexose Transporters 1 (TbTHT1), and human GLUT3, both of which are membrane sugar transporters. NGT was localized by fluorescence microscopy to subcellular structures consistent with lysosomes. Silencing NGT expression with RNA interference in parasites resulted in a growth defect …

Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage

Masters Theses

Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a …

Illuminating Transfer Rna Variants As Genetic Modifiers In Models Of Human Disease, Jeremy T. Lant

Electronic Thesis and Dissertation Repository

Transfer RNAs (tRNAs) physically link the genetic code to an amino acid sequence, by recruiting amino acids to three-nucleotide codons in messenger RNAs. To ensure that the genetic code is translated as intended, tRNAs must be accurately aminoacylated and faithfully recognize codons in the ribosome during protein synthesis. Given the critical function of tRNAs, it has often been assumed that mutations in human tRNA genes would be either lethal to cells or not significantly impair tRNA function. My goal was to rigorously test this assumption in mammalian cell models, prompted by the recent discovery of unprecedented variation in human tRNA …

Genetic Analysis Of Adhesion Protein Elmo3 In Arabidopsis Thaliana, Garrison Asper

Honors Projects

The Extracellular Matrix (ECM) between plant cells is vital for structure, development, and intercellular adhesion. A pectin rich layer in between cells, the middle lamella, is largely responsible for regulating the adhesive properties of adjacent plant cells. Homogalacturonan (HG) pectin, the most common, is synthesized in the Golgi and secreted into the ECM where it undergoes calcium crosslinking, increasing its adhesive properties. Mutations in proteins essential for HG synthesis can reveal a severe adhesion defective phenotype, where the hypocotyls of dark grown Arabidopsis exhibit cell sloughing, curling, and general disorganization. A family of five ELMO proteins are suspected to act …

Functional Characterization Of Cancer-Associated Dna Polymerase Ε Variants, Stephanie R. Barbari

Theses & Dissertations

Replicative DNA polymerases ε (Polε) and δ (Polδ) achieve high fidelity DNA synthesis through a precise balance of polymerization and exonucleolytic proofreading. Errors that escape proofreading are corrected by DNA mismatch repair (MMR). Ultramutated human cancers with proficient MMR carry alterations in the exonuclease domain of Polε, which were initially predicted to abolish proofreading. However, functional studies in yeast of the most recurrent Polε-P286R variant suggested defects beyond a loss of exonuclease activity. Indeed, biochemical analysis of the yeast Polε-P286R analog revealed increased polymerization capacity in addition to decreased proofreading, which enables efficient mismatch extension and bypass of replication-blocking non-B …

Erecta Family Genes Regulate The Shoot Apical Meristem And Organ Formation, Daniel A. Degennaro

Doctoral Dissertations

Plants are sessile and must adjust their organ growth to their environments. A reservoir of stem cells in the shoot apical meristem (SAM) supplies cells for differentiation into organs. The SAM must balance organ production with stem cell maintenance. The ERECTA family (ERfs) encodes the leucine-rich repeat receptor-like kinases ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERL2. ERf signaling regulates organ initiation and stem cell maintenance. Results presented in this work include the following:

1) WUSCHEL (WUS) and CLAVATA3 (CLV3) make up a negative feedback loop to maintain SAM size. WUS and CLV3 expression localization is critical for …

C. Elegans Response To Cadmium Toxicity, Brian James Earley

Arts & Sciences Electronic Theses and Dissertations

Cadmium is an environmental pollutant and significant health hazard that is similar to the physiological metal zinc. Residing in the same group of the periodic table, cadmium and zinc share chemical characteristics that are important for their industrial uses in electroplating, batteries, pigments, and metal alloys. The similarities of ionic cadmium and zinc have significant repercussions on biological systems. While it has long been clear that cadmium is toxic to biological systems, the mechanisms of cadmium toxicity remain poorly understood. In contrast, mechanisms of zinc homeostasis have been elucidated in growing detail. In C. elegans high zinc homeostasis is regulated …

Discovery Of Novel Ubiquitin- And Methylation-Dependent Interactions Using Protein Domain Microarrays, Jianji Chen

Dissertations & Theses (Open Access)

Post-translational modifications (PTMs) drive signal transduction by interacting with "reader" proteins. Protein domain microarray is a high throughput platform to identify novel readers for PTMs. In this dissertation, I applied two protein domain microarrays identifying novel readers for histone H2Aub1 and H2Bub1, and H3TM K4me3. Ubiquitinations of histone H2A at K119 (H2Aub1) and histone H2B at K120 (H2Bub1) function in distinct transcription regulation and DNA damage repair pathways, likely mediated by specific "reader" proteins. There are only two H2Aub1-specific readers identified and no known H2Bub1-specific readers. Using a ubiquitin-binding domain microarray, I discovered the phospholipase A2-activating protein (PLAA) PFU domain …

Functional Characterization Of Threonine 49 Phosphorylation Of Cytochrome C, Antoine Khobeir

Wayne State University Theses

Cytochrome c (Cytc) is a pivotal multifunctional mitochondrial protein that serves as a single electron carrier between complexes III and IV of the electron transport chain. It has important roles in both cellular respiration and apoptosis. The novel Thr49 (T49) phosphorylation of Cytc likely affects mitochondrial respiration, membrane potential, ROS production, ATP production, and apoptosis. Based on the functional characterization of previously mapped phosphorylation sites (Tyr97, Tyr48, Thr28, Ser47, Thr58) of the lab, we hypothesize that T49 phosphorylation will lead to controlled respiration, optimal intermediate mitochondrial membrane potential, lower ROS production, and inhibition of apoptosis compared to unphosphorylated Cytc. Here …

Sars-Cov-2: An Investigation On Mutagenicity And Its Effects On Infectivity And Mortality, Tyler Elliott Silverwood

Honors Theses and Capstones

SARS-CoV-2, the etiological agent of the COVID-19 pandemic, has rapidly become a worldwide public health concern. Classified as a betacoronavirus, it is the third human coronavirus (HCoV) to emerge in the 21st century that causes severe disease, alongside SARS-CoV and MERS-CoV. The genome consists of open reading frames encoding accessory proteins and four structural proteins, including the spike protein which is a key determinant of host cell tropism. Mutations within the genome, particularly the spike gene, have been linked in-vitro to increased binding affinity to the human receptor angiotensin-converting enzyme 2 (hACE2), increased fitness in human hosts, and immune evasion. …

Mnrr1: Understanding The Role Of A Novel Mitochondrial-Nuclear Regulator, Stephanie L. Gladyck

Wayne State University Dissertations

Mitochondria are complex organelles that generate most of the energy required to sustain life and function in metabolic and signaling pathways required to maintain cellular homeostasis. MNRR1 (mitochondrial nuclear retrograde regulator 1 or CHCHD2) is a small, bi-organellar twin CX9C protein that is emerging as an important regulator of mitochondrial function, apoptosis, and cellular stress by participating in mitochondrial-nuclear crosstalk. Our lab has previously shown that in the mitochondria, MNRR1 regulates complex IV (Cytochrome c oxidase or COX) and is able to finetune the oxidase function through phosphorylation status. We have also shown that during stress, mitochondrial MNRR1 levels deplete, …

Genetic Analysis Of Cellular Adhesion In Arabidopsis Thaliana, Andrew Close Bolender

Honors Projects

Plant cell adhesion is mediated by the extracellular matrix (ECM) or cell wall and plays an important role in plant morphogenesis and development. The amount, modification, and cleavage of pectin in the cell wall are major contributors to the adhesive properties of the ECM. To gain a more complete picture of plant cell adhesion processes, Arabidopsis thaliana seedlings were previously mutagenized and screened for hypocotyl adhesion defects. Genomic sequencing of one plant exhibiting an adhesion defect, isolate 242, showed that two mutations, one in cellulose synthase (CesA1) and another in a sugar transporter, are candidates for the causative mutation. This …

A Molecular Toolset For The In Vivo Detection Of A Sulfolobus Islandicus Leucyl Trna Synthetase Paralog, Nicholas Michael Bretz

Theses and Dissertations

An ancient and ubiquitous set of enzymes known as the aminoacyl-tRNA synthetases are required for the viability of all organisms. The aminoacyl-tRNA synthetases catalyze the attachment of amino acids onto tRNA molecules. The aminoacylated, or charged, tRNA is then transported and utilized at the ribosome for the synthesis of proteins. The genome of the hyperthermophilic microorganism Sulfolobus islandicus (S. islandicus) harbors a unique leucyl-tRNA synthetase paralog, LeuRS-I, of unknown function. While its duplicate, LeuRS-F, carries out all the classical functions of LeuRS-family enzymes, LeuRS-I fails to charge leucine onto tRNALeu, despite its ability to activate this amino acid and bind …

Artificial Intron Technology To Generate Conditional Knock-Out Mice, Amber N. Thomas-Gordon

Dissertations & Theses (Open Access)

Genetic engineering has been re-shaped by the invention of new tools in modern biotechnology in a way that offers precision and efficiency in modifying the genome at a single nucleotide level and/or allowing precise control of gene expression. Such gene manipulation brings about significant findings and revelations in comprehending more about embryonic development, cellular and physiological functions, and disease pathology. Current methods used to produce conditional knockouts have limitations on conditional allele placement and modification varies among genes in different organisms. Thus, a system for generating conditional alleles with fidelity remains a challenge. My goal was to examine an approach …

Understanding Cytochrome C Maturation In Anaerobic Archaea, Blake Wojciechowski

Biological Sciences Undergraduate Honors Theses

Methanoperedens nitroreducens (MPEBLZ), an archaeal methanotroph and close relative of Methanosarcina acetivorans, contain numerous cytochrome c proteins. However, difficulty in using these organisms as a model for cytochrome cresearch has created a pressure to express cytochrome c proteins in an organism that is much easier to work with. A punitive cytochrome c protein (MPEBLZ_04274) from M. nitroreducens was attempted to be cloned into a model methanogen M. acetivorans as well as Escherichia coli. Cytochrome c proteins are important for many metabolic processes within anaerobic archaea. In order for a mature cytochrome c to be formed heme must …

Generation Of Conditional Ku70 Knockouts In Human Cell Lines Using Crispr/Cas9 And Dual-Nuclease Crispr/Tevcas9, Gursimran Parmar

Electronic Thesis and Dissertation Repository

The Ku heterodimer, Ku70 and Ku80, plays a key role in DNA repair. Viable Ku70 knockouts exist in mice but not in human cell lines. The objective was to create Ku70 knockouts and evaluate knockout viability in human cells using CRISPR/Cas9 and TevCas9. Editing is achieved by Cas9 through one sequence-specific blunt cut accompanied by error-prone DNA repair. However, TevCas9, a novel fusion protein of Cas9 and I-Tev, creates two non-compatible DNA breaks and biases editing events towards a small deletion. Ku70 has five processed pseudogenes therefore intron-exon junctions were targeted by gRNA and a cell line stably transfected with …

Role Of Microrna-483 In Pancreatic Β-Cells, Jackson Waugh

Dissertations, Master's Theses and Master's Reports

Insulin is an essential hormone produced by β-cells in the pancreas. The release of insulin is tightly regulated in healthy people in order to control blood sugar level in our body. However, people with Type 2 Diabetes have insufficient insulin secretion from pancreatic β-cells, leaving to high blood sugar (hyperglycemia) and β-cell failure. microRNAs (miRNAs or miR) are newly discovered small regulatory molecules and have emerged as important regulator of cell growth, differentiation, and organ function. Altered miRNA function has been implicated in the pathogenesis of a variety of human disease, including diabetes. In this report, we focus on dissecting …

Epigenetic Implications In Inorganic Arsenic-Mediated Carcinogenesis, Meredith Eckstein

Theses and Dissertations--Molecular and Cellular Biochemistry

Chronic, low dose exposure to inorganic arsenic (iAs) is a public health concern throughout the world, contributing to the development of many diseases, including lung cancer. Several mechanisms for iAs-mediated carcinogenesis have been proposed, of which the production of reactive oxygen species and formation of chromosomal aberrations are the most studied. Another equally important, yet less studied mechanism is dysregulation of epigenetic marks. “Epigenetics” refers to changes that occur on the DNA and chromatin that do not alter base pair identity, but alter compaction, expression, and regulation of specific DNA sequences. There are several types of epigenetic marks including histone …

The Exploration Of Nanotoxicological Copper And Interspecific Saccharomyces Hybrids, Matthew Joseph Winans Phd

Graduate Theses, Dissertations, and Problem Reports

Nanotechnology takes advantage of cellular biology’s natural nanoscale operations by interacting with biomolecules differently than soluble or bulk materials, often altering normal cellular processes such as metabolism or growth. To gain a better understanding of how copper nanoparticles hybridized on cellulose fibers called carboxymethyl cellulose (CMC) affected growth of Saccharomyces cerevisiae, the mechanisms of toxicity were explored. Multiple methodologies covering genetics, proteomics, metallomics, and metabolomics were used during this investigation. The work that lead to this dissertation discovered that these cellulosic copper nanoparticles had a unique toxicity compared to copper. Further investigation suggested a possible ionic or molecular mimicry …

Activation And Regulation Of The Alkbh3-Ascc Alkylation Repair Pathway, Josh Brickner

Arts & Sciences Electronic Theses and Dissertations

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions. These lesions are sensed by distinct pathways to recruit repair factors specific to type of damage. In particular, the ALKBH family of proteins recognizes and repairs specific alkylated lesions, including 1-methyladenine (m1A) and 3-methylcytosine (m3C). A major outstanding question in the field is how the AlkB homologue ALKBH3 and its associated protein partners are recruited to sites of alkylation damage and how this repair activity is regulated. Understanding the upstream signaling events that mediate recognition and repair of DNA alkylation damage is particularly …

Mechanisms Of Trinucleotide Repeat Instability During Dna Synthesis, Kara Y. Chan

Theses and Dissertations--Toxicology and Cancer Biology

Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a …

Temperature And Polyunsaturated Fatty Acid’S Effect On Daphnia Magna Reproduction, Mark Albright

Electronic Theses and Dissertations

Organisms adapt to their environments by adjusting their biochemistry and physiology; such adaptation is limited by resource availability and physiological constraints. The freshwater crustacean Daphnia magna inhabits a wide range of environments and must survive and reproduce within a range of temperatures. One limit to low-temperature adaptation is thought to be the availability of unsaturated fatty acids necessary to maintain proper fluidity of cellular membranes. D. magna maintained at 10 ºC on a diet poor in unsaturated fatty acids have been observed to produce clutches that fail to develop. However, this has not been observed on a diet rich in …