Molecular Genetics Commons^™

Utilizing Crispr/Cas9 Gene Editing Determine The Role Of Cnr1 During Zebrafish Development, Camme Sells

[Archive] Belmont University Research Symposium (BURS)

In a world where cannabinoids are being used more frequently as for medicinal purposes, the importance for research on cannabinoid biology is steadily increasing. The goal of this study was to gain a greater understanding of the effect cannabinoids have on developing zebrafish embryos. Work in this area of developmental biology could give way to an understanding of the safety of a human fetus during pregnancy with a mother who uses cannabis medicinal therapy. The CRISPR/Cas 9 system was utilized in this study to genetically edit the zebrafish embryo genome to remove the function of the Cannabinoid Receptor 1 ( …

Enhancement Of Nkx3.2 Expression After Twist1b And Twist2 Knockdown In Zebrafish, Peyton Yearick

[Archive] Belmont University Research Symposium (BURS)

Somitogenesis is a developmental event patterned by cellular signaling during gastrulation, ending around 30-hours post fertilization. Somites are segmented regions of mesoderm along the dorsal side of the body which form paraxial mesoderm in Danio rerio, zebrafish. Somite development results in the formation of the subcompartment called sclerotome, which differentiates to form the axial skeleton and associated tendons (Stickney et al., 2000). Further differentiation allows for sclerotome progenitor cell to develop into bone, muscle, tendon, and cartilage through sclerotome migration along the mesoderm. A portion of sclerotome cells migrate anterior towards the neural tube, where they will form the …

Expression Of Tgf-Beta Ligands During Sclerotome Development, Estela Williams

[Archive] Belmont University Research Symposium (BURS)

During early embryogenesis in vertebrates, somites are formed by the segmentation of the presomitic mesoderm (Gomez, Ozbudak, Wunderlich, Baumann. Lewis and Pourquie 2008). The somite possesses several domains within it and the ventral part of the somite is called the sclerotome. The sclerotome gives rise to the axial skeleton and tendons. Twist 1b and Twist 2 are genes that are expressed in the sclerotome during axial skeletal development. Our experiments were developed to help understand the roles of Twist1b and Twist2 in tendon development. By utilizing microinjection to alter gene expression, the Twist 1b and Twist 2 genes can be …

Using Crispr/Cas9 Gene Editing To Remove Twist2 To Determine Its Role During Somite Development In Zebrafish, Caleb Holdener

[Archive] Belmont University Research Symposium (BURS)

Embryonic Mesoderm is a precursor to various types of tissues, such as the somatic mesoderm. Somites are a segmented tissue that gives rise to the sclerotome in vertebrates. This sclerotome further develops into the axial skeleton (Stickney, Barresi, and Devoto 2000). Somites are easily observable under low power microscopy during fish development. In fish, great strides have been made in studying the developmental fate of somites (Stoiber, Haslett, and Sänger 1999; Stickney, Barresi, and Devoto 2000). Zebrafish have been studied in many labs due to their similarities with higher vertebrates. These advantages include their inexpensive cost of care, external fertilization, …

Twist1b Knockout Generation In Zebrafish Using Crispr-Cas9, Grace Hurley

[Archive] Belmont University Research Symposium (BURS)

The Twist gene is found in numerous organisms, including humans, mice and zebrafish (Yeo et al., 2009). It is a vital gene in development and is needed for the organism to survive (Yeo et al., 2009). Specifically, the Twist gene family aids in specialization and differentiation of mesodermal cells, telling somites, segmented blocks of mesoderm, what to become (Germanguz et al., 2007). Over time, the Twist gene has duplicated into paralogs, each having differences in their functions (Germanguz et al., 2007). Twist1b is a protein coding gene that is a paralog to the human Twist1 gene (ZFIN, 2020). In zebrafish, …

Scleraxis Expression Within The Axial Tendon Population In Zebrafish, Jessica Bernaba

[Archive] Belmont University Research Symposium (BURS)

Zebrafish are a well-established model organism for vertebrate development. During embryonic development, a pattern of segmentation of mesoderm yields somites, which give rise to specific cell fates. Somites, bilateral blocks of mesoderm along the neural tube in developing vertebrate embryos, subdivide into sclerotome as the vertebrate matures. Depending on migratory position, sclerotome cells give rise to various components of the axial skeletal system, including bone, cartilage, and tendon. Each fate is determined by gene expressions within the sclerotome and occupy distinct domains of the body plan. By method of in situ hybridization, we studied the expression of the scleraxis ( …

Utilizing Crispr/Cas9 Gene Editing Determine The Role Of Cnr1 During Zebrafish Development, Ellis Chase

[Archive] Belmont University Research Symposium (BURS)

The CRISPR-Cas 9 system is a process used to create mutations, insertions, or deletions within a desired gene. It can be used to create mutant organisms necessary to study the function and impact of a particular gene¹. Cannabinoids are active chemicals found in cannabis, the most common of which being cannabinol (CBD)². Cannabinoid receptors are an integral part of the endocannabinoid system, recognizing cannabinoid signals to promote physiological processes like pain sensation, memory, mood, and appetite³. Studying these cannabinoid receptors can provide insight into how cannabis can affect bodily processes.

Zebrafish are small translucent …

Exploring The Role Of Tgfbr2 During Zebrafish Sclerotome Development, Nooralhuda Feron

[Archive] Belmont University Research Symposium (BURS)

Zebrafish, Danio rerio, are used as a source model organism for human development of the tendons, bones, and ligaments. The sclerotome is an embryonic structure known as the somite. The sclerotome gives rise to axial bones and tendons. During development, different genes are involved and affect how these skeletal tissues develop. Specifically, within this project, the effects of Twist1b and Twist2 were studied to see their effects on tendon formation, as they are expressed within the developing sclerotome tissue. Transforming growth factor beta receptor type-2 (Tgfbr2) has been shown to be involved in tendon formation (Subramanian, Kanzaki, Galloway and …

Environmental Dna Metabarcoding Detects Mammal Use Of Stock Tanks And Natural Springs On The Prescott National Forest, K. A. Smith, Berenice Carreras Mendiolea, C. E. Benson, F. B. Anaya

Discovery Day - Prescott

Recent research has shown the viability of environmental DNA (eDNA) analysis as a tool for identifying species presence in aquatic ecosystems. However, there is limited research indicating the feasibility of eDNA as a survey tool in water sources such as springs and stock tanks. Because of the scarcity of water in the high desert landscape of Arizona, natural springs and stock tanks are an important water resource. In this study, we examined whether 16S rRNA metabarcoding could detect mammal eDNA in springs and stock tanks in the Prescott National Forest. During summer 2019, we collected and analyzed water samples at …

Relative Model Fit Does Not Predict Topological Accuracy In Single-Gene Protein Phylogenetics, Stephanie J. Spielman

College of Science & Mathematics Departmental Research

It is regarded as best practice in phylogenetic reconstruction to perform relative model selection to determine an appropriate evolutionary model for the data. This procedure ranks a set of candidate models according to their goodness of fit to the data, commonly using an information theoretic criterion. Users then specify the best-ranking model for inference. Although it is often assumed that better-fitting models translate to increase accuracy, recent studies have shown that the specific model employed may not substantially affect inferences. We examine whether there is a systematic relationship between relative model fit and topological inference accuracy in protein phylogenetics, using …

Diversity, Function And Regulation Of Cell Surface And Intracellular Immune Receptors In Solanaceae, Jong Hum Kim, Christian Castroverde

Biology Faculty Publications

The first layer of the plant immune system comprises plasma membrane-localized receptor proteins and intracellular receptors of the nucleotide-binding leucine-rich repeat protein superfamily. Together, these immune receptors act as a network of surveillance machines in recognizing extracellular and intracellular pathogen invasion-derived molecules, ranging from conserved structural epitopes to virulence-promoting effectors. Successful pathogen recognition leads to physiological and molecular changes in the host plants, which are critical for counteracting and defending against biotic attack. A breadth of significant insights and conceptual advances have been derived from decades of research in various model plant species regarding the structural complexity, functional diversity and …

An Intergrative Ecological And Taxonomic Assessment Of The Stoneflies (Insecta, Plecoptera) Of The Black Mountains, North Carolina, Usa, Madeline Lake Metzger

Masters Theses & Specialist Projects

The Black Mountains are a part of the Blue Ridge region of the Appalachian Mountains and include Mount Mitchell, the highest peak east of the Mississippi River at 2,037 m (= 6,684 ft). A comprehensive inventory of the stonefly fauna was conducted between 2014-2019 within Mt. Mitchell State Park and adjacent Pisgah National Forest. Collections occurred seasonally from 44 unique locations, covering a broad range of stream sizes and elevations in the region. Sequencing of the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was completed to assist with making positive determinations to species for females of taxonomically challenging genera …

Diversity Of The Major Histocompatibility Complex In African Penguins (Spheniscus Demersus) In Situ, Athena Schalk

Undergraduate Honors Thesis Projects

The Major Histocompatibility Complex (MHC) is responsible for the immune response in all jawed vertebrates and protects individuals against a variety of pathogens and diseases. Maintaining genetic diversity within the MHC exons is critical to protecting endangered species. African penguins (Spheniscus demersus) are in danger of losing their MHC diversity in isolated populations due to reductions in population size associated with environmental changes and human activity. This study analyzes the diversity within the exons in the DNA encoding the MHC by amplifying the exons through polymerase chain reaction and identifying alleles through denaturing gradient gel electrophoresis. Wild populations of …

Tip60 Regulation Of Δnp63Α Is Associated With Cisplatin Resistance, Akshay Hira, Andrew Stacy, Jin Zhang, Michael P. Craig, Madhavi Kadakia

Celebration of Undergraduate & Graduate Research, Scholarship, and Creative Activities Materials

About 5.4 million basal and squamous cell skin cancers are diagnosed every year in the US. ΔNp63a, a member of the p53 transcription factor family, is overexpressed in non-melanoma skin cancer and regulates cell survival, migration and invasion. TIP60 is histone acetyltransferase (HAT) which mediates cellular processes such as transcription and the DNA damage response (DDR). Previous studies in our lab have shown that overexpression of TIP60 induces ΔNp63a protein stabilization in a catalytic-dependent manner. Since ΔNp63a is known to transcriptionally regulate several DDR genes and promote cisplatin resistance, its stabilization by TIP60 may contribute to the failure of platinum-based …

Assessing Seed Handling Processes To Facilitate A Community-Engaged Approach To Regional Forest Restoration, Meredith Cobb, Michaela J. Woods, Ryan Mcewan

Biology Faculty Publications

In the Midwestern United States, there is a strong management impetus toward reforestation to replace trees lost to due to tree mortality and to establish forests on previously cleared properties. Here, we describe a public outreach and volunteer effort that aimed to generate positive, community-engaged restoration outcomes and report on a practical experiment involving seed handling. We obtained tree seeds that were donated by members of the community to local land managers. We evaluated the size of the seeds collected and tested the influence of common stratification media (none, sand and vermiculite) and seed size on germination success of three …

Association Of Interleukin 17f With Arthritis In Punjabi Families Of Pakistan, Asia Parveen, Andleeb Batool, Maryam Mukhtar, Abdul Wajid, Naila Malkani

Journal of Bioresource Management

Arthritis is a chronic inflammatory disease that causes severe joint pain. Interleukin 17F (IL17F) is considered as a candidate gene functionally; it mediates pro-inflammatory responses, depending on the type and site of inflammation. The present study examined the polymorphism of IL17F (rs763780 and rs2397084) among the families affected by arthritis. Demographic data and blood samples were collected from the families with at least one affected offspring with arthritis. Analysis of the IL17F gene polymorphism was performed by the digestion of DNA with NlaIII and AvaII. The results showed that IL17F rs763780 (AA, AG and GG genotypes) and rs2397084 (AA, AG …

Functions Of Cdk/Cyclin Complexes In Endoreplication Regulation By The Cdk Inhibitor Siamese, Kai Wang

LSU Doctoral Dissertations

Arabidopsis trichome (leaf hair) is a specialized single cell extended from epidermal cell on the leaves, which is a typical endoreplication and is also known as endoreduplication. Several D-type cyclins were tested to check the cell division in trichome, and the trichome expressing either CYCB1;2 or CDKB2;2 cannot trigger cell division, even if simultaneous expression of CYCB1;2 and CDKB2;2 failed to produce mitosis in trichome. Only CYCD3;1 specifically promotes multicellular trichome. cdkb1;1cdbk1;2 double mutants and sim cdkb1;1cdkb1;2 triple mutants exhibit the phenotype similar to the wild type and very limited cell division respectively. Overexpression of a CDKB1;1 dominant-negative construct that …

Molecular Differentiation Of Astragalus Species And Varieties From The Western United States: The Chloroplast Dna Bridge Between Evolution And Molecular Systematics, Marwa Neyaz, Daniel Cook, Rebecca Creamer

Poisonous Plant Research (PPR)

Locoweeds are the most widespread poisonous plant problem in the world and have been reported in the Western United States since the 1800s, causing tremendous losses in livestock. Consumption of locoweeds by grazing animals stimulates the neurological disease, locoism, characterized by weight loss, ataxia, and lack of muscular coordination. The name locoweed is used for Astragalus and Oxytropis species known to contain swainsonine, the toxic principle produced by the plant endophytic fungus Undifilum. Astragalus includes 2,500-3,000 species and many varieties that have almost identical morphological characteristics that overlap among species, leading to improper identification. Therefore, the aim of this study …

A Positive Feedback Loop Of Hippo- And C-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration, Madison Irwin, Meghana Tare, Aditi Singh, Oorvashi Roy Puli, Neha Gogia, Matthew Riccetti, Prajakta Deshpande, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Alzheimer's disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a Drosophila eye model of AD by targeted misexpression of human Aβ42 in the differentiating retinal neurons, where an accumulation of Aβ42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly …

The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis Via Oxidative Damage, Holly A. Martin, Amanda A. Kidman, Jillian Socea, Carmen Vallin, Mario Pedraza-Reyes, Eduardo A. Robleto

Life Sciences Faculty Research

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the …

Tamalin/Gras-1 Connects Glutamate Receptor Activity To The Insulin/Igf Signaling Cascade To Regulate Neuroprotection In A Nematode Model Of Excitotoxicity, Ayesha Chowdhury

Dissertations, Theses, and Capstone Projects

Brain ischemia is a major cause of debilitation and death in the United States. Excitotoxicity, a condition that arises from the accumulation of glutamate (Glu) in the synapse that leads to overactivation of Glu receptors (GluRs), is the major mechanism of neuronal damage in brain ischemia / stroke. Although it is commonly acknowledged that over activation of GluRs leads to neurodegeneration, it has been recently shown that even during excitotoxicity Glu has a concurrent important role in regulating neuroprotection. GluR-activated transcription factors seem to mediate this neuroprotection, but it remains unclear which signaling cascades and transcription factors are regulated by …

Identification Of Uncommon Antibiotic-Producing Illinois Soil Isolates, Lesly Muniz, Dr. Lori Scott

Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil

This project is a collaboration with the Tiny Earth Project Initiative (TEPI), which is a global network of educators and students focused on student sourcing antibiotic discovery from the soil. We researched tester strains B. subtilis and E. coli from the soil isolates obtained. We further verified if the isolates were common antibiotic bacteria. Unfortunately, this project heavily relied on biochemical tests, colony morphology, and Gram stains to reject or fail to reject our hypothesis. Our goal was to discover new antibiotic-producing bacteria that could be beneficial in combating ESKAPE strains. A proper PCR and DNA extraction would be required …

Pseudomonas And Bacillus Soil Isolates Produce Antibiotics, Chelsea Brandt, Dr. Lori Scott

Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil

The recent emergence of antibiotic resistance bacterial strains presents a significant challenge and threat to human healthcare. While new methods of treatment such as bacteriophage therapy and combinations of existing antibiotics are being researched, the human population is in dire need of new antibiotics to replace those that are ineffective. This research addresses this need by identifying antibiotic producing bacteria in a soil sample from Davenport, IA. This project is a collaboration with the Tiny Earth Project Initiative (TEPI), which is a global network of educators and students focused on studentsourcing antibiotic discovery from soil. Microbiology lab techniques and 16S …

Identification Of Antibiotic Producing Soil Bacteria Against Bacillus Subtilis, Morgan Brockhouse, Dr. Lori Scott

Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil

This project is a collaboration with the Tiny Earth Project Initiative (TEPI), which is a global network of educators and students focused on student-sourcing antibiotic discovery from soil. Individual strains of soil bacteria were isolated and produced antibiotic against Bacillus subtilis. Two of these samples were sequenced using the 16S rRNA gene to reveal they are very closely related to the genus Pseudomonas.

Gene Expression Pattern Analysis Of Anterior Hox Genes During Zebrafish (Danio Rerio) Embryonic Development Reveals Divergent Expression Patterns From Other Teleosts, Adam Davis

Georgia Journal of Science

The regional identity of organs and organ systems along the anterior-posterior axis during embryonic development is patterned, in part, by Hox genes, which encode transcription factor proteins that activate or repress the expression of downstream target genes. Divergent nested Hox gene expression patterns may have had a role in facilitating morphological divergence of structures, such as the pharyngeal jaw apparatus, among evolutionarily divergent teleost fishes. Recent studies from several evolutionarily divergent teleosts, such as the Japanese Medaka (Oryzias latipes) and the Nile Tilapia (Oreochromis niloticus), have shown the presence of divergent expression patterns of several Hox …

Frontotemporal Dementia Nonsense Mutation Of Progranulin Rescued By Aminoglycosides, Lisha Kuang, Kei Hashimoto, Eric J. Huang, Matthew S. Gentry, Haining Zhu

Molecular and Cellular Biochemistry Faculty Publications

Frontotemporal dementia (FTD) is an early onset dementia and is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD is highly heritable with mutations in progranulin accounting for 5-26% of cases in different populations. Progranulin is involved in endocytosis, secretion and lysosomal processes, but its function under physiological and pathological conditions remains to be defined. Many FTD-causing nonsense progranulin mutations contain a premature termination codon (PTC), thus progranulin haploinsufficiency has been proposed as a major disease mechanism. Currently, there is no effective FTD treatment or therapy.

Aminoglycosides are a class of antibiotics that possess a less known function …

Using Crispr-Cas9 As A Restriction Enzyme, Zack Crawley

Master's Theses

Restriction digests are a commonly utilized process for cleaving DNA at specific, but relatively common sites. Restriction enzymes have widespread use in DNA manipulation. CRISPR/Cas9 is a recently identified endonuclease which utilizes a customizable guide sequence to recognize and cut specific ~20 bp sites located in a DNA sequence. This preliminary research aimed to exploit the potential benefit of DNA restriction using the CRISPR/Cas9 procedure through alterations of different components involved in that system. We sought to refine existing CRISPR/Cas9 protocols and make a budget friendly, user-selectable CRISPR/Cas9 restriction digest protocol. The motivation for this research was to simplify and …

The Physical Interactions Of Klp-4 & The Effects Of Microtubule Acetylation, Lindsey Wright

Honors Program Theses

Our nervous system propagates signals by releasing neurotransmitters into the synaptic cleft between two adjacent neurons. The transport and delivery of such neurotransmitters within the neuron is reliant on microtubule structure and motor proteins present. The direction of motor protein transport is dictated by the inherent polarity of microtubules. Kinesin-3 motor proteins, such as KLP-4 found in Caenorhabditis elegans, use this polarity to travel in an anterograde direction towards neurotransmitter exocytosis sites. Thus, KLP-4 is associated with the maintenance of neuron functionality. The klp-4 (ok3537) allele results in a large, in frame deletion within the cargo-binding domain of the motor …

Single Nucleotide Polymorphism (Snps) Study On X-Chromosome In Thai Sle Populations, Krisana Jaiwan

Chulalongkorn University Theses and Dissertations (Chula ETD)

Systemic Lupus Erythematosus (SLE) is common autoimmune disease in Thailand which dominantly in females in ratio 9:1 of patients. Previous study has shown that the genetic components especially in X chromosome contributed a lot to the disease development. However, the susceptibility loci in Thai population have not been fully examined. Here, we conducted genome-wide association study (GWAS) on X chromosome using the data from two independent cohorts: primary dataset (controls = 1,683, SLE = 487) and secondary dataset (controls = 1,711, SLE = 455). Through meta-analyzing and imputation base on 1 KGP the two data set, SNP rs1059702 in IRAK1- …

The Origin And Evolution Of Flower Characters In Angiosperms With A Case Study On The Genetic Basis Of Zygomorphic Corolla Development In Bee And Hummingbird Pollinated Species Of Schizanthus (Solanaceae), Ghadeer Bukhari

Theses and Dissertations

This Ph.D. dissertation investigates two main projects separated into two chapters, one for each. The first chapter explores the evolution of flowering plants (angiosperms), a macro-scale study. The second project is on a micro-scale that focus on exploring the genes that regulate the similarities and the differences of flower morphology reflecting their different pollination syndromes in two species of Schizanthus (Solanaceae), i.e., S. pinnatus, a bee-pollinated flower, and S. grahamii, a hummingbird- pollinated flower.

In the first chapter, I used floral diagrams to study the origin and the evolution of floral traits in angiosperms, a new set of morphological data …