Molecular Genetics Commons^™

Optimizing The Expression Of Polyethylene Terephtalate Hydrolase-Encoding Synthetic Gene In Escherichia Coli Arctic Express (De3), Jocelyn Nataniel, Maria Ulfah, Dini Achnafani, Niknik Nurhayati, Gabriela Christy Sabbathini, Sri Rezeki Wulandari, Abinawanto Abinawanto, Is Helianti

Makara Journal of Science

The waste of polyethylene terephthalate (PET) plastic waste in Indonesia is a pressing concern due to its slow degradation and potential environmental damage. One promising solution is to utilize polyethylene terephthalate hydrolase from Ideonella sakaiensis (IsPETase), an enzyme that specifically degrades PET. However, inducing the expression of IsPETase synthetic gene in Escherichia coli BL21 (DE3) has been challenging because much of it remains insoluble. This study aimed to express IsPETase in E. coli Arctic Express (DE3) and optimize the conditions to enhance its production. First, pET22b(+)pelB-IsPETase was inserted into E. coli Arctic Express (DE3). The …

Characterization Of Ato Family Transporters In The Fungal Pathogen Cryptococcus Neoformans, Will Betsill

All Theses

Fungal pathogens are a significant threat to public health as they are becoming increasingly common and more resistant to treatment. Cryptococcus neoformans contributes greatly to this threat annually by causing an estimated 278,000 cases of cryptococcal meningitis resulting in approximately 181,000 deaths globally according to the CDC. C. neoformans is ubiquitous across most of the globe and can be found in such places as in trees or soil. Exposure to this fungus is especially dangerous to individuals who are immunocompromised or immunosuppressed. In these cases, inhalation of spores can lead to infection in the lungs. Once in the lungs, C. …

Characterizing The Role Of Pa5189 Of Pseudomonas Aeruginosa In Deletion And Overexpression Mutants, Seh Na Mellick

Theses/Capstones/Creative Projects

In the context of rising multidrug resistance in biofilm-forming pathogens like Pseudomonas aeruginosa, this study investigates the role of the understudied transcription factor PA5189 in antibiotic resistance and biofilm formation. PA5189 deletion and overexpression mutants were created in a parent P. aeruginosa strain using pEX18Tc-based recombinant suicide vectors, with genotypic verification of putative triparental conjugants achieved through restriction digestion and PCR. The study revealed that PA5189 overexpression significantly increases resistance to commonly used broad spectrum antibiotics such as ciprofloxacin and imipenem. Additionally, differential expression of PA5189 was found to notably affect biofilm formation, with variations contingent on the nutrient …

Trna Anticodon Cleavage By Target-Activated Crispr-Cas13a Effector, Ishita Jain, Matvey Kolesnik, Konstantin Kuznedelov, Leonid Minakhin, Natalia Morozova, Anna Shiriaeva, Alexandr Kirillov, Sofia Medvedeva, Alexei Livenskyi, Laura Kazieva, Kira S Makarova, Eugene V Koonin, Sergei Borukhov, Konstantin Severinov, Ekaterina Semenova

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA–guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus …

The Plant Disease Triangle Facing Climate Change: A Molecular Perspective, Charles Roussin-Léveillée, Christina A. M. Rossi, Christian Castroverde, Peter Moffett

Biology Faculty Publications

No abstract provided.

Exploring The Role Of Calcineurin In Filamentation Across Inducing Conditions In The Fungal Pathogen Candida Albicans, Patricia Harte-Maxwell, Jill R. Blankenship

UNO Student Research and Creative Activity Fair

Candida albicans is an opportunistic human pathogenic fungus affecting millions of people globally; high mortality rates are possible in systemic infection. Filamentation or a filamentous growth pattern is a primary virulence mechanism of C. albicans underlying other virulent behaviour....

Managing Stress: A Study Of Stress Response Mechanisms In Mycobacteria, Augusto C. Hunt Serracin

Biology Dissertations - Archive

Mycobacteria encompass many pathogenic species known to cause severe disease in humans. A well-known example is Mycobacterium tuberculosis (Mtb), the causative agent of the lung disease tuberculosis, which kills millions of humans worldwide yearly. Pathogenic mycobacteria like Mtb are challenging to treat because of their innate ability to adapt to environmental stress. Their unique cell physiology and conserved stress responses allow them to combat biological insults, regulate growth, and regulate genes involved in stress; all these responses increase tolerance to antibiotics. The current therapies to treat mycobacterial infections are lengthy and, at times, unsuccessful, partly due to antibiotic tolerance. A …

Contributions Of The Replication Initiator Protein Dnaa To Physiology And Virulence Of The Lyme Disease Spirochete, Andrew Krusenstjerna

Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics

Borrelia burgdorferi, the etiological agent of Lyme disease, is the most prevalent vector-borne disease in the United States. The spirochetal population is naturally maintained by consistently cycling between Ixodid ticks and small vertebrates. To survive within these diverse niches, B. burgdorferi has evolved a unique physiology and regulatory network to sense and respond to environmental fluxes. Replication is a hallmark of the Lyme spirochete’s enzootic life cycle, specifically at the nymphal tick-to-vertebrate interface. The mechanisms and regulatory schema for the basic cellular processes in B. burgdorferi, or indeed any member of the phylum spirochaetota, are largely unknown. The central hypothesis …

Ribosome Hibernation Factor Induces Antibiotic Persisters Of Mycobacterium Tuberculosis In Neutrophil-Rich Lung Lesions, Jamie Corro

Electronic Theses & Dissertations (2024 - present)

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is a prescient global health threat and the leading cause of death by an infectious agent. Treatment of Mtb infection involves administering 2-4 antibiotics for a minimum of 6 months. This extended drug regimen is required to target a subpopulation of persistent bacilli. As their name implies, these bacteria can “persist” by acquiring nonhereditary and transient antibiotic tolerance. Although the precise mechanism is unknown, it’s often attributed to slowing cellular growth and metabolic processes. During zinc deprivation, bacteria can remodel their ribosomes by replacing their zinc-binding paralogues containing the CXXC motif (C+) …

Investigating Birds As Dispersal Vectors Of Litylenchus Crenatae Subsp. Mccannii (Anguinidae), The Nematode Associated With Beech Leaf Disease, Spencer Rock Parkinson

Graduate Theses, Dissertations, and Problem Reports (ETD)

Beech leaf disease (BLD) is an emerging forest pathogen primarily affecting American beech (Fagus grandifolia, Ehrh.) in North America and has been attributed to tree mortality of sapling sized trees within five to seven years of infection. Symptoms typically occur in regenerating American beech thickets sprouting from roots of trees killed by beech bark disease. Scientists first observed BLD in Ohio in 2012 and currently has spread to 15 states in the USA and one Canadian province. The nematode Litylenchus crenatae subsp. mccannii (Lcm) is highly associated with BLD symptoms, interveinal chlorosis and defoliation of leaves, and is currently …

Tim/Tam Receptors: A Potential Biomarker For Predicting Sensitivity To Zika Virus-Induced Oncolysis In Non-Small Cell Lung Cancers, Shankari Somasekar

Honors Undergraduate Theses

Non-small cell lung cancers (NSCLC) constitute 80-85% of lung cancers and are the leading cause of cancer-related deaths globally. The most common cause is prolonged smoking. Current treatment options for NSCLC include surgery, radiation, chemotherapy, targeted drug therapy, and immunotherapy. Although these medications are effective in the short term, patients often face issues of drug resistance and debilitating side effects with prolonged use. Currently, the use of Zika virus (ZIKV) is being researched as a possible alternative treatment for cancer, which minimizes side effects and the risk of drug resistance. TIM/TAM proteins are identified as the putative ZIKV receptors on …

Consequences Of Spatial Structure In Soil–Geomorphic Data On The Results Of Machine Learning Models, Daehyun Kim, Insang Song, Lorrayne Miralha, Daniel R. Hirmas, Ryan W. Mcewan, Tom G. Mueller, Pavel Samonil

Biology Faculty Publications

In this paper, we examined the degree to which inherent spatial structure in soil properties influences the outcomes of machine learning (ML) approaches to predicting soil spatial variability. We compared the performances of four ML algorithms (support vector machine, artificial neural network, random forest, and random forest for spatial data) against two non-ML algorithms (ordinary least squares regression and spatial filtering regression). None of the ML algorithms produced residuals that had lower mean values or were less autocorrelated over space compared with the non-ML approaches. We recommend the use of random forest when a soil variable of interest is weakly …

Increased Coding Potential Of Bovine Herpesvirus 1, Victoria Jefferson

Theses and Dissertations

Bovine respiratory disease (BRD) costs the cattle industry millions of dollars in costs in treatment and loss every year in the United States. A significant pathogen often contributes to BRD is Bovine Herpesvirus 1 (BoHV-1), a double stranded DNA virus with the ability to establish latency in the trigeminal ganglia and neurons. Primary infection with BoHV-1 results in immunosuppression that increases the risk of secondary bacterial infection and pneumonia. Because herpesviruses infect their hosts for life and can be reactivated in times of stress, BoHV-1 can present a recurring risk of BRD. The following research aims to expand the knowledge …

Identification And Characterization Of Geotrichum Candidum Isolates From Peach With Reduced Sensitivity To Propiconazole, Sara Price

All Theses

Geotrichum candidum Link (1809) is a yeast-like fungus that causes sour rot of stone fruits, including peach (Prunus persica). The disease caused damage between 2021 and 2023 to the peach crop kept in cold storage at a commercial farm in South Carolina despite preharvest and postharvest applications of fungicides. Isolates from 41 symptomatic fruit, from a South Carolina (SC) commercial packing house, as well as other isolates obtained from South Carolina were identified to be G. candidum based on species-specific PCR amplifications of the β tubulin gene. Commonly used postharvest fungicide fludioxonil had no effect on G. candidum mycelial growth …

Characterizing The Regulation Of Candida Albicans Hyphal Morphogenesis In The Context Of Host Macrophages, Hannah Wilson

Dissertations and Theses (Open Access)

Candida albicans is a commensal fungus that resides on the skin, mucosal surfaces, and in the gut of an estimated 80% of individuals. Though generally harmless, C. albicans is capable of causing uncomfortable mucosal infections as well as deadly disseminated infections depending on the immune status of the host. Although antifungal therapeutics exist, the mortality rate associated with disseminated disease still lingers around 50%. Further, C. albicans is the fourth most common cause of all bloodstream infections and continues to present as a major clinical issue with less than satisfactory treatment options. For this reason, it is imperative to understand …

Modeling Nonsegmented Negative-Strand Rna Virus (Nnsv) Transcription With Ejective Polymerase Collisions And Biased Diffusion, Felipe-Andres Piedra

Research Symposium

Background: The textbook model of NNSV transcription predicts a gene expression gradient. However, multiple studies show non-gradient gene expression patterns or data inconsistent with a simple gradient. Regarding the latter, several studies show a dramatic decrease in gene expression over the last two genes of the respiratory syncytial virus (RSV) genome (a highly studied NNSV). The textbook model cannot explain these phenomena.

Methods: Computational models of RSV and vesicular stomatitis virus (VSV – another highly studied NNSV) transcription were written in the Python programming language using the Scientific Python Development Environment. The model code is freely available on GitHub: …

Transcriptional Pausing Factor M1bp Regulates Cellular Homeostasis By Suppressing Autophagy And Apoptosis In Drosophila Eye, Anuradha Venkatakrishnan Chimata, Hannah Darnell, Akanksha Raj, Madhuri Kango-Singh

Biology Faculty Publications

During organogenesis cellular homeostasis plays a crucial role in patterning and growth. The role of promoter proximal pausing of RNA polymerase II, which regulates transcription of several developmental genes by GAGA factor or Motif 1 Binding Protein (M1BP), has not been fully understood in cellular homeostasis. Earlier, we reported that M1BP, a functional homolog of ZKSCAN3, regulates wingless (wg) and caspase-dependent cell death (apoptosis) in the Drosophila eye. Further, blocking apoptosis does not fully rescue the M1BP^RNAi phenotype of reduced eye. Therefore, we looked for other possible mechanism(s). In a forward genetic screen, members of the Jun-amino-terminal-(NH2)-Kinase (JNK) pathway …

Investigating Transcriptional Anomalies During Persistence In Chlamydia Trachomatis, Nathan D. Hatch

Theses & Dissertations

Chlamydia trachomatis (Ctr) is the leading cause of bacterial sexually transmitted infections. Ctr, a Gram-negative, obligate intracellular bacterium, develops within a membrane-bound vacuole termed an inclusion. This protective, nutrient rich, intracellular environment has allowed Ctr to undergo extensive reductive evolution – resulting in a streamlined genome of approximately 1 Mbp and the loss or fragmentation of several biosynthetic pathways, including the ability to synthesize tryptophan (Trp) de novo. During infection, the host immune system releases the cytokine IFNγ. Upon binding to a host cell, IFNγ stimulates the expression of indoleamine-2,3-dioxygenase, an enzyme capable of catabolizing cytosolic Trp, thereby starving …

Spatial Structure Formation By The Post-Transcriptional Gene Regulator Rsme In Pseudomonas Fluorescens Pf0-1, Anton Evans

Electronic Theses and Dissertations

Microorganisms are often found in microbial communities we call biofilms. Organisms living in these crowded environments have significant evolutionary pressure to retain access to the resources necessary to sustain life. My research uses the bacterium Pseudomonas fluorescens Pf0-1 to study how organisms evolve strategies to solve this crowding problem as aging colonies repeatedly generate mutant patches. These mutants expand the reach of the colony resulting in decreased local density as they push themselves up to the resource rich surface. These spatial structures result from social interactions between the mutant and the parental cells mediated through extracellular secretions, resulting in the …

Sensing The Rhizosphere: Microbial Transcriptome Responses To Root Exudates And Redox-Active Metabolites, Ankita Bhattacharyya

Dissertations

The term “rhizosphere” describes the dynamic interface between plant roots and soil influenced by root exudates. It is a hotspot of microbial activity, plant-microbe, and microbe-microbe interactions. Distinct variations in bacterial diversity are thought to be driven by plant root exudates. However, the molecular details of these processes still remain unclear.

I addressed this gap by focusing on two strains, Pseudomonas synxantha 2-79 and Burkholderia lata 383, representing diverse groups of Gammaproteobacteria comprised of species with environmental, agricultural, and medical significance. In the first part of this project, we used a combination of metabolomics and RNA-seq to characterize transcriptome responses …

Molecular Mechanisms Behind Sars-Cov-2 Induced Host Genome Restructuring, Erin Simpson

Dissertations and Theses (Open Access)

Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) has caused unprecedented morbidity and mortality worldwide. There are two pathophysiological hallmarks associated with severe patient outcomes after SARS-CoV-2 acute infection, namely delayed/weakened interferon production and overactive inflammatory responses. Our previous work has shown that these altered immune responses are due, at least in part, to significant alterations in 3D genome organization and epigenetic landscape following SARS-CoV-2 infection that are distinct from and more severe than changes caused by other viral infections or immune stimulants. While these changes at the chromatin level are important for understanding the immune dysregulation seen in some cases …

The Role Of Adenovirus Serotype 5 E4 11k In The Relocalization Of The Cellular P Body Proteins, Kasey A. Karen, La`Quita Randolph, Kevin Neubrecht, Heather Vincent

Georgia Journal of Science

Human adenoviruses are a useful tool to understand basic cellular biology in addition to viral infections. Historically, cellular splicing was first discovered in adenovirus, but other cellular processes, such as double-strand break repair and aggresome formation, have been further elucidated through adenoviral infection. The adenovirus protein E4 11k has been shown to disrupt cytoplasmic processing bodies (p bodies), which are not well-understood but are involved in mRNA metabolism. Several p body proteins were found to be reorganized in the cytoplasm with adenovirus serotype 5 (Ad5) able to cause the colocalization of these p body proteins with aggresomes. The p body …

N-Acetyltransferase 9 Ameliorates Aβ42-Mediated Neurodegeneration In The Drosophila Eye, Prajakta Deshpande, Anuradha Venkatakrishnan Chimata, Emily E. Snider, Aditi Singh, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, manifests as accumulation of amyloid-beta-42 (Aβ42) plaques and intracellular accumulation of neurofibrillary tangles (NFTs) that results in microtubule destabilization. Targeted expression of human Aβ42 (GMR > Aβ42) in developing Drosophila eye retinal neurons results in Aβ42 plaque(s) and mimics AD-like extensive neurodegeneration. However, there remains a gap in our understanding of the underlying mechanism(s) for Aβ42-mediated neurodegeneration. To address this gap in information, we conducted a forward genetic screen, and identified N-acetyltransferase 9 (Mnat9) as a genetic modifier of GMR > Aβ42 neurodegenerative phenotype. Mnat9 is known to stabilize microtubules by inhibiting c-Jun-N- …

Overexpressing Two Helicobacter Pylori Small Rnas From A Bacterial Pathogenicity-Related Chromosomal Region To Investigate Their Regulation Of Virulence Genes, Roxanne N. Mcpeck, Olivia F. Morgan, Andrea R. Castillo Phd

2023 Symposium

The bacterial pathogen Helicobacter pylori infects the stomachs of approximately 50% of humanity, causing symptomatic disease (e.g., stomach ulcers, gastric cancer, and MALT lymphoma) in 10-15% of the infected. Colonizing the acidic, inhospitable stomach requires H. pylori to tightly regulate gene expression despite lacking many common bacterial genetic regulatory elements. The pathogen may compensate by using abundant non-protein-coding small RNAs (sRNAs) to regulate gene expression, including of infection-intensifying virulence genes. Additionally, severe disease and cancer correlate with infection by H. pylori strains that contain a nonessential chromosomal region, the cytotoxin-associated gene pathogenicity island (cagPAI). This encodes powerful virulence …

Elucidating The Impact Of Sos-Response Timing In On Escherichia Coli Survival Following Treatment With Fluoroquinolone Topoisomerase Inhibitors, Stephanie Schofield

Honors Scholar Theses

Antibiotic treatment failure is a public health crisis, with a 2019 report stating that roughly 35,000 deaths occur in the United States yearly due to bacterial infections that are unresponsive to antibiotics (1). One complication in the treatment of bacterial infection is antibiotic persistence which further compromises our battle to effectively treat infection. Bacterial persisters can exist in clonal bacterial cultures and can tolerate antibiotic treatment by undergoing reversible phenotypic changes. They can survive drug concentrations that their genetically identical kin cannot. Some persisters remain in a slow growing state and are difficult to target with current antibiotics. A specific …

Understanding The Role Of Heterodisulfide Reductase During Nitrogen Fixation By Methanosarcina Acetivorans, Grant Shelnut

Biological Sciences Undergraduate Honors Theses

Methanosarcina acetivorans is a methanogenic archaeon (methanogen) that is important for several processes in our biosphere. A feature of interest within this methanogen is the enzyme nitrogenase which catalyzes the reduction of unusable dinitrogen gas (N₂) into a usable form of nitrogen, ammonia (NH₃). How electrons are transferred within M. acetivorans to nitrogenase during N₂ reduction is unknown. Heterodisulfide reductase (Hdr) is a key enzyme known to reduce the electron carrier protein ferredoxin, which is a direct electron donor to nitrogenase in bacteria. I hypothesized that cytoplasmic HdrA2B2C2 functions in electron transfer to nitrogenase and …

Gene Expression Changes Linked To Phenylpropanoid-Based Resistance To Fusarium Head Blight Of Wheat, Shiv Singla

Department of Plant Pathology: Dissertations, Theses, and Student Research

Fusarium graminearum is a devastating pathogen of wheat that causes Fusarium head blight (FHB) and contaminates the grain with the mycotoxin deoxynivalenol (DON). Resistance to FHB is quantitative and it is important to identify additional genes conferring resistance against it. The goal of this thesis was to examine if the constitutive expression of two sorghum phenylpropanoid pathway genes, SbCCoAOMT (encoding caffeoyl-CoA O-methyltransferase) and SbC3’H (encoding p-coumarate 3-hydroxylase), in the moderately-susceptible spring wheat CB037 can provide Type-I and Type-II resistance to F. graminearum and determine the underlying mechanisms of the enhanced resistance. The constitutive expression lines (CCoAOMT413, CCoAOMT421, C3H112, …

Functions Of The Trna Splicing Endonuclease And Other Adventures In Rna Processing, Jennifer Hurtig, Ambro Van Hoof

Dissertations and Theses (Open Access)

The tRNA splicing endonuclease (TSEN), has been studied for over three decades for its function in tRNA splicing. However, this enzyme has other functions that are just beginning to be characterized. Mutations in TSEN cause the neuronal disease pontocerebellar hypoplasia (PCH) that is characterized by atrophy of the cerebellum and pons, overall developmental failure, and usually results in death before adolescence. How mutations in TSEN cause these neuronal defects and disease is not understood. In yeast, TSEN has another essential function that is independent of tRNA splicing and is still unknown. In this thesis I strived to understand the other …

Diagnosis Of Urinary Tract Infections And Rapid Molecular Characterization Of Antibiotic Resistance, Mohammed Harris

All Dissertations

Urinary tract infections (UTIs) are one of the most common infectious clinical entities in both community and hospital settings. They have a broad range of clinical severity yet inflict large epidemiological burden of morbidity and mortality on patients and the healthcare system with billions of dollars in cost of treatment. Understanding what methods are optimal for diagnosing UTIs are critical to mitigate the marked impact and cost of these infections.

Chapter 1 and 2 in this work surveys the broad array of diagnostic modalities for UTIs and highlights their advantages and limitations in the context of the current standard of …

Pyrus Calleryana Extracts Reduce Germination Of Native Grassland Species, Suggesting The Potential For Allelopathic Effects During Ecological Invasion, Michaela J. Woods, Jonathan T. Bauer, Dena Schaeffer, Ryan W. Mcewan

Biology Faculty Publications

Invasive plant species’ success may be a result of allelopathy, or the release of secondary metabolites that are harmful for surrounding plant species. Allelopathy can be mediated through the abiotic environment by chemical sorption or transformation, so the substrate on which interactions occur can lead to differential outcomes in allelopathic potential. One aggressive invader, Pyrus calleryana, has become dominant in many ecosystems throughout Eastern US, and has reduced the abundance of native species where it invades. Thus, our goal was to identify if P. calleryana had allelopathic potential by testing the impact of leaf and flower leachate on gemination …