Molecular Genetics Commons^™

Determining Genes Involved In Recovery From Chemotherapy In 4t1, Emt6, And Eo771 Breast Cancer Cells, Akash Jagdeesh, Joseph Landry Ph.D.

UROP Posters

Breast cancer is one of the most common types of cancer, and often has poor prognosis. Traditional treatments for breast cancer include chemotherapy, which involves a chemical attack on all growing cells within the body. However, these treatments are not 100% effective, and patients might experience recurrence in their cancer months or years after achieving remission. Cancer cells can evade chemotherapy through five mechanisms: senescence, quiescence, cytoprotective autophagy, and apoptosis/necroptosis suppression. Prior studies have conducted CRISPR screens and experiments involving inhibition of epigenetic regulators, to alter the regulation of genes that can contribute to breast cancer chemotherapy resistance. This study …

Fluorescent Protein Imaging Approaches Uncover Dynamic Subcellular Distribution Of Core Clock Proteins In Neurospora Crassa, Ziyan Wang

Dartmouth College Ph.D Dissertations

Circadian clocks are endogenous self-sustained timekeeping systems that allow organisms to anticipate and adapt to daily environmental cycles. Found in diverse forms of life, these molecular clocks regulate the expression of ~ 40% of the genome in animals, fungi, and plants. In animals and fungi, circadian oscillators are built on transcription-translation feedback loops (TTFLs), in which positive regulators drive the expression of negative regulators which, once synthesized and imported into the nucleus, inhibit their own activators. This precisely regulated negative feedback incorporates multiple steps of delays that are essential for generating ~24-hour rhythms. Through these mechanisms, circadian clocks temporally coordinate …

Building A Cell Specific System For In Vivo Rna Imaging Of The Endogenous Actin Gene In C. Elegans, Jasmine A. Huerta Canete

Theses and Dissertations

Messenger RNA (mRNA) plays a crucial role in protein synthesis and cellular processes, important for the development of cells such as neurons. When dysregulation occurs, it can lead to outcomes such as aberrant translation, causing neurodegenerative conditions to arise. Unfortunately, the localization of mRNA in an in vivo cell specific manner is still not understood. To address this, we decided to develop a cell-specific system to image RNA in vivo, within the organism known as C. elegans with a focus on the actin gene. The Actin gene encodes for an important cytoskeleton protein expressed ubiquitously, vital for the cell …

Bio-Orthogonal Chemistry-Based Strategy To Turn-On And Turn-Off Crispr-Cas9 Gene Editing In Solution And In Live Cells, Bhoomika Pandit

Electronic Theses & Dissertations (2024 - present)

The CRISPR–Cas9 system is a widely popular tool for genome engineering. There is a strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this thesis I describe two biorthogonal based approaches to either Turn-ON and Turn-OFF CRISPR-Cas9 gene editing.

In first project work I describe a bio-orthogonal chemistry-based approach to Turn-ON Cas9 nuclease activity with temporal precision. We report a TCO-acylimidazole reagent that acylates 2′-OH groups of RNA. Poly-acylation (“cloaking”) of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well …

Mechanisms Of Glyphosate Toxicity: Uptake, Mitochondrial Function, And Metabolism, Dionysios Patriarcheas

Graduate Theses, Dissertations, and Problem Reports (ETD)

Glyphosate, the most widely used herbicide in the world, is an inhibitor of chorismate biosynthesis, the precursor of aromatic amino acids. The target of glyphosate is absent in animals, but findings reporting toxicity in model organisms without a known mechanism or target are increasing. Saccharomyces cerevisiae is a useful and versatile model for understanding molecular mechanisms of toxicity. In yeast, previous work has shown that the aspartate/glutamate permease Dip5 is the proposed transporter for glyphosate and that mitochondrial metabolism has been implicated in response to glyphosate toxicity. This dissertation aimed to identify the effects of glyphosate on mitochondrial function through …

Genetics Of Mycangial Development And Host Colonization Within The Ambrosia Symbiosis, Mckeon James Laws

Graduate Theses, Dissertations, and Problem Reports (ETD)

Ambrosia beetles, a polyphyletic group of weevils, utilize pouch-like structures at various locations on the body to transport for their sole food source, their fungal partner. Ambrosia fungi are known to originate from multiple unrelated fungal lineages with no consensus of traits required to become a fungal cultivar. While the biology and ecology of both partners is in many cases well understood, the genetic underpinnings of this fascinating mutualism remain a mystery. The aim of this dissertation is to add to the understanding of the ambrosia symbiosis by providing insights into its molecular and genetic underpinnings through RNA sequencing data, …

Phylogeography, Fungal Host Specificity, And Fitness In The Corallorhiza Maculata Species Complex, Hana Thixton-Nolan

Graduate Theses, Dissertations, and Problem Reports (ETD)

Mycoheterotrophic plants exploit fungi for most or all nutritional needs, providing a compelling system to address the evolutionary consequences of genome reduction, population-level variation, and germination requirements. I am using the widespread Corallorhiza maculata species complex to address questions of genomic variation, fungal host specificity, morphological variation, and germination requirements across the broad geographic range from Mexico to northern North America. This complex contains C. bulbosa, C. macrantha, and C. mertensiana, the latter with three varieties (maculata, occidentalis, and mexicana). These taxa vary in flowering time, floral morphology, and fungal associations. However, the degree …

Mechanistic Insight Of Neuropilin-2 Expression During The Progression Of Prostate Cancer, Sanika Bodas

Theses & Dissertations

Advanced prostate cancer represents a particularly aggressive and challenging form of prostate cancer (PCa) that shows resistance to both traditional therapies, such as androgen deprivation therapy, and newer treatment modalities like hormone therapies and immunotherapies. For patients diagnosed with advanced stages of PCa, treatment options are limited, and chemotherapy is often the last line of defense. Unfortunately, chemotherapy can severely diminish the quality of life, particularly in older patients, due to its harsh side effects, including fatigue, immune suppression, and overall physical decline. Despite these challenges, researchers and clinicians remain focused on developing more effective and targeted treatments for patients …

Regulation Of Chromatin Remodeling By The Post-Hsa Domain Of Brg1, A Subunit The Baf Atp-Dependent Chromatin Remodeling Complex, Min Sze Ewe

Theses & Dissertations

The ATP-dependent chromatin remodeler BAF complex regulates nucleosome positioning, chromatin accessibility, and tissue specific gene expression. Its catalytic subunit, BRG1, contains several regulatory domains including the post-HSA region, proposed to modulate ATPase activity. Multiple cancer-associated mutations map to these regulatory regions underscoring their importance, yet the precise role of the post-HSA region in chromatin remodeling remains poorly understood.

To investigate the function of the post-HSA domain, we ectopically expressed FLAG-tagged BRG1 with post-HSA deletion (Δpost-HSA BRG1) in mouse embryonic stem cells. Western blot analysis confirmed expression of the mutant protein. To determine the effects of mutant protein expression on chromatin …

Generating Predictive Gene Expression Signatures For Alzheimer's Disease Using Postmortem Brain Tissue, Ashley Duche

Pharmaceutical Sciences (PhD) Dissertations

Background: Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid-beta (Aβ) plaques and tau protein aggregates. These pathological features develop in specific brain regions, but why some areas are more vulnerable to early AD-related changes remains unclear. To address this, predictive gene expression signatures were developed to explore the molecular mechanisms underlying regional susceptibility to AD pathology.

Methods: This was performed using postmortem brain (PMB) tissue from participants in the Religious Orders Study and Memory and Aging Project (ROSMAP), Mayo Clinic, and Mount Sinai Brain Bank (MSBB) to generate gene expression signatures from six brain …

Structure And Function Studies On Trna Splicing Endonuclease, Justin G. Underwood

Dissertations and Theses (Open Access)

A subset of eukaryotic pre-tRNAs have introns that must be spliced out to become mature molecules capable of functioning in translation. This essential processing step is performed by tRNA splicing endonuclease (TSEN), a highly conserved heterotetramer with two catalytic and two structural subunits. In addition to this conserved function, archaeal TSEN also processes pre-rRNA and yeast TSEN cleaves some mRNAs that encode mitochondrial proteins to initiate mRNA degradation. Interestingly, single amino acid mutations in TSEN cause the human Mendelian disease pontocerebellar hypoplasia (PCH) by an unknown mechanism. Recently, several groups have determined the structure of human TSEN, revealing similarities and …

Neural Rrp6 Depletion Drives Circrna Accumulation And Motor Impairment In Drosophila Melanogaster Neurons, Joseph Serio

Honors Theses

Circular RNAs (circRNAs) are a class of ubiquitous eukaryotic RNAs created by back splicing. These noncoding RNAs can modulate gene expression and are implicated in aging and neurodegenerative diseases. To limit the contribution of circRNAs to aging and pathobiology, it is essential to understand how turnover of these molecules is regulated, as it is not clear which enzymes are chiefly responsible for degrading circRNAs in neurons. This study evaluates the roles of ribonucleases potentially involved in neural circRNA turnover using the model fly Drosophila melanogaster. A mifepristone-activated GAL4/UAS GeneSwitch expressed RNAi transgenes and inactivated the ribonucleases. Flies were evaluated on …

Improved Model For Neurodegeneration In C. Elegans, Jean-Pierre Arditi

Graduate Theses and Dissertations (2019 - present)

During Alzheimer's disease (AD), insoluble amyloid beta (AP) peptides accumulate to form extracellular aggregates (plaques). The direct cause of neuronal dysfunction observed in AD has been broadly investigated. The amyloid hypothesis states that AP plaques are neurotoxic, but recent studies support the AP oligomer hypothesis, which states that intracellular AP (iAP) is neurotoxic. To test this hypothesis, I used CRISPR-Cas9 gene editing to generate two transgenic Caenorhabditis elegans (C. elegans) strains. I generated a strain (RSLl 11) using the rab-3 promoter to co-express GFP in neurons, which showed no gross behavioral changes but had a 50% reduced egglaying rate. RSLl …

Fostering Entrepreneurial Thinking And Practice Through Teaching And Learning Integration, Mohammad G. Sabbir

Learning and Educational Center Virtual Teaching and Learning Conference

This session explores an innovative, evidence-based approach to integrating entrepreneurial thinking into undergraduate neuroscience education through hands-on gene editing experiences. Using the commercially available Bio-Rad CRISPR/Cas9-based “Out of the Blue” kit for bacterial LacZ gene editing, students are guided not only through the scientific principles underlying modern gene editing technologies but also through the entrepreneurial challenge of replicating and manufacturing the kit using existing lab resources. This model empowers students to extend their learning beyond the classroom by engaging in cost-effective, scalable biotechnology practices—particularly valuable for institutions with limited funding. Classroom-based research demonstrates increased student engagement, deeper conceptual understanding, and …

Bioaccumulation Pattern Of Per- And Polyfluoroalkyl Substances (Pfas) In Fish Tissues From Two Freshwater Systems, Margaret D. Taiwo, Husam Kafeenah, David D. Duvernell, Michael O. Eze

Biological Sciences Faculty Research & Creative Works

Per- and polyfluoroalkyl substances (PFAS) are known for their persistence, ubiquity, bioaccumulation in different matrices of the environment and their detrimental effect on human health. In this study, we used EPA 1633 to examine the prevalence of ten PFAS compounds in two freshwater systems and investigated their bioaccumulation pattern across different tissues of grass carp fish (Ctenopharyngodon idella), common carp (Cyprinus carpio), and flathead catfish (Pylodictis olivaris). Among the PFAS compounds analyzed, PFBS exhibited the highest concentration in the freshwater sample, exceeding the U.S. EPA regulatory limit of 4 ng/L for drinking water. The total PFAS concentrations in the muscle, …

Genetic Characterization And High-Resolution Total Mrna Sequencing Of The Millipede Cherokia Georgiana Bollman, 1889, Elena Cruz

Biology Theses

There are approximately 12,000 described species within the class Diplopoda. Only six species, falling within four of sixteen orders, have fully sequenced genomes. No whole genomes are available for incredibly diverse families like Xystodesmidae. Many interesting characteristics in this group are poorly defined at the genetic level, such as the production of a defensive hydrogen cyanide secretion and UV fluorescence in the order Polydesmida. Here, we present a genetic characterization of the polydesmid millipede Cherokia georgiana Bollman, 1889. We include tissue-specific sequencing metrics, alignment and assembly of mitochondrial DNA consensus sequence according to tissue type, and phylogenetic tree construction using …

Purple Tomatoes Boost Nutrition Crop Value And Create New Opportunities For United States (U.S) Agriculture, Tariq Alam

Agronomic Crops

The agricultural landscape in the United States is continually evolving, with growers and the tomato industry seeking innovative ways to meet consumer demands while enhancing profitability. The introduction of both bioengineered and classically bred anthocyanins-enriched purple tomatoes presents a unique opportunity for U.S. growers to cultivate a high-value crop that appeals to niche markets and commands premium pricing. Bioengineered purple tomatoes achieve high anthocyanin levels through the introduction of snapdragon transcription factors, while classically bred 'Indigo Rose' purple tomatoes are developed via classical breeding methods, providing an alternative for consumers who prefer traditionally bred products. Specifically, the purple tomato holds …

Evaluating The Potential And Limitations Of Nanopore Adaptive Sampling For Targeted Transcriptome Sequencing, Nicole Debruyne, Feng Wang, Yang Xu, Lan Lin

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Long-read RNA sequencing is a powerful technology for transcriptomics, but low throughput and high cost pose challenges. Adaptive sampling, a feature of Oxford Nanopore Technologies, offers real-time enrichment by selectively ejecting non-target molecules. We evaluate adaptive sampling for human transcriptome analysis. Adaptive sampling modestly enriches target transcripts (1.3 × for cDNA sequencing, 1.9 × for direct RNA sequencing) while preserving gene expression and splicing profiles, but is significantly less effective than cDNA hybridization capture. Short read lengths and low sequencing quality limit performance. Adaptive sampling on direct RNA sequencing can boost target yield (~ 20%) within fixed run times, potentially …

Bioinformatic Analysis Of Pogz Variants In Relation To White Sutton Syndrome, Hannah Rollins

Theses

White-Sutton syndrome (WHSUS) is a rare neurodevelopmental disorder caused by mutations in the Pogo Transposable Element with ZNF Domain (POGZ) gene, which encodes pogo-transposable element with ZNF domain, a chromatin regulator essential for proper mitotic progression and DNA repair. This study uses a bioinformatic framework to evaluate the structural and functional impact of missense mutations in the conserved amino acid region (positions 500–800) of the POGZ protein. Protein modeling, variant effect prediction, conservation analysis, and molecular dynamics simulations were employed to gain an understanding of the effects of POGZ missense mutations on protein structure and movement with specific emphasis on …

Investigating The Presence Of R-Loops At The Centromeres Of Drosophila Melanogaster, Daniel D'Souza

Holster Scholar Projects

This study investigates the presence and localization of R-loops at the centromeres of Drosophila melanogaster. R-loops are non-canonical nucleic acid structures that have been implicated in various cellular processes, including genomic instability and accurate chromosome segregation. Although previous research has found R-loops at Drosophila satellite sequences and LTR retrotransposons, their presence and function at the centromeres of a whole organism remain largely unknown. Using IF-FISH (immunofluorescence-fluorescence in situ hybridization), we stained R-loops with the S9.6 antibody. A UAS-rnh1 overexpression line was used to induce the overexpression of RNase H1, an enzyme that resolves R-loops, to confirm the specificity of …