Molecular Biology Commons^™

In Vitro Reversal Of Abc Transporter Mediated Multidrug Resistance In Human Ovarian And Non-Small Cell Lung Cancer Models, Alison K. Kellom, Pia D. Vogel

Biological Sciences Theses and Dissertations

One of the major causes of treatment failure in aggressive cancers is multidrug resistance (MDR), which is linked to the overexpression of membrane efflux proteins that export chemotherapeutics from cancer cells. This mechanism prevents chemotherapeutic drugs from reaching cytotoxic concentrations intracellularly, allowing the cancer to survive. Two primary mediators of this mechanism are P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). P-gp and BCRP are transmembrane ATP-binding cassette (ABC) transporters that are frequently overexpressed in MDR cancers. They utilize the binding and hydrolysis of ATP to transport a diverse range of amphipathic molecules of varying size (Schinkel and Jonker, 2003), …

Transcriptomic Study On The Effect Of Salt Stress In Sorghum Bicolor Seedlings, Joshwa A. Gandy

Theses and Dissertations

The global impact and reliance on cereal crops, and sorghum in particular, is paramount for worldwide food security and for the livelihood of hundreds of millions of people. The detrimental effects of salt stress on crop plants and the huge economic losses it causes by affecting agricultural output worldwide is a devastating issue with huge impacts, which have prompted research to combat salinity in numerous ways. Understanding how cereals respond to the negative effects of salt stress is crucial for enhancing their salt tolerance. In this study of sorghum, we used a 3’-end RNA sequencing approach to elucidate major salt …

Implications Of Acetylation And Phosphorylation On Metabolic Enzymes, Nour Fatema

Graduate Theses and Dissertations

All living things depend on glycolysis and the TCA cycle for energy production and metabolic control. Post-translational modifications (PTMs) such as acetylation and phosphorylation, which can change enzyme activity, substrate binding, and overall metabolic balance, tightly regulate these pathways. Diseases like cancer and metabolic problems are associated with disruptions in these alterations.

Serine phosphorylation and lysine acetylation are two of the many PTMs that are evolutionarily conserved processes that alter protein function. Previously thought to be a static histone alteration, acetylation is now understood to be a dynamic regulator of metabolic enzymes that modifies contacts, charge, and conformation. In response …

Examining The Biotic And Abiotic Degradation Of Simple Phenols, Madeline Gruenberg

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Plant secondary metabolites are a diverse class of compounds that encompass small phenols to large polymeric structures such as tannins. Smaller compounds such as phenolic acids can serve as substrates for soil microbes and enzymes. We monitored the degradation of three small phenols pyrogallol, gallic acid, and benzoic acid in biotic and abiotic conditions. They were chosen to observe the impacts of specific functional groups on both degradation processes and act as model compounds to mimic larger tannin reactivity. Both abiotic and biotic degradation of pyrogallol resulted in the formation of a stable quinone, while abiotic degradation of gallic acid …

The Role Of Fbxl16 In Regulating Erk3 Stability And Cell Migration, Seth Charles Murdock

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Extracellular signal-regulated kinase 3 (ERK3) is a member of the atypical mitogen-activated protein kinase (MAPK) family. While ERK3’s role in promoting cell migration and invasion in multiple types of cancer has been well-documented, its activation and regulation remain poorly understood. Unlike other typical MAPK proteins, whose activation is regulated via the phosphorylation of an upstream kinase, ERK3 is regulated through its abundance, as it is constitutively phosphorylated when present in the cell. While USP20 has been established as a deubiquitinating protein that stabilizes ERK3 by preventing its degradation, both TRIM21 and FBXW7 have been identified as E3 ligases that target …

Regulation And Function Of Mec-2 Alternative Splicing In Single Cells, Canyon Calovich-Benne

Biological Sciences Theses and Dissertations

One mechanism for generating diversity at the single cell level is selective expression of a unique set of transcription factors (TFs) and RNA binding proteins (RBPs). These regulatory factors contribute to functions of cells by controlling cell fate, turning genes on and off, and processing of transcripts either co- or post-transcriptionally. Many genes, especially neuronal genes, have multiple different splicing events, promoters, and/or polyadenylation sites. Post-transcriptional coordination is in the infancy of being studied and the functional consequences of this coupling at the single cell level has yet to be explored. Here we show, mec-2 coordination of alternative transcription start …

Unveiling The Nexus Of Cellular Quality Control: Exploring The Interplay Between Ribosome-Associated Protein Quality Control And Mitochondrial Quality Control Pathways, Foozhan Tahmasebinia

Biological Sciences Theses and Dissertations

In eukaryotic cells, the intricate interplay between cellular quality control mechanisms is crucial for maintaining homeostasis and safeguarding the integrity of vital processes, spanning from macromolecule synthesis to the renewal of entire cellular organelles.

Disruption of these networks can lead to severe diseases such as metabolic disorders, underscoring the interconnected nature and feedback control mechanisms inherent in biological systems, including cellular quality control systems. This interconnectedness extends to the intricate communication between organelles, enabling coordinated functioning and adaptation to changing cellular conditions, particularly in response to stressors.

While the exact mechanisms governing these communications within cellular quality control systems remain …

Long-Read Mdm4 Isoform Sequencing Reveals Aberrant Isoform Landscape In Metastatic Melanomas, Nehaal Rooha Patrick

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The changes in splicing of MDM4 in human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Genomic characteristics of melanoma and precursor lesions, such as copy number variation and gene expression, have been investigated in detail. However, there has never been a detailed survey of splicing changes that occur during melanomagenesis. Splicing changes can be a very early event in melanoma tumor progression, with characteristic changes in the p53 pathway already in place in early nevi. MDM4 is upregulated in a strong majority of melanoma cases and has been described as a “key therapeutic target in cutaneous …

Exploring The Effects Of Choline Treatment On Dystrophic Phenotype, Brooklyn Morris

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Duchenne Muscular Dystrophy (DMD) is a devastating progressive muscular disorder caused by a mutation in the dystrophin gene affecting approximately 1 in 3,500 males. However, despite its prevalence, there is currently no cure for this disease. Mutations in dystrophin lead to enhanced inflammation, fibrosis, cell death, development, and decreased skeletal muscle function. Phosphatidylcholine, synthesized by choline, is a major phospholipid that functions in maintaining and synthesizing cell membranes and has previously been found to be decreased in DMD patients. This led us to hypothesize that the dystrophic phenotype could be improved through treatment with choline. In this study, we used …

Profiling And Verifying The Substrates Of E3 Ubiquitin Ligase Rsp5 In Yeast Cells, Shuai Fang, Geng Chen, Yiyang Wang, Rakhee Ganti, Tatiana A Chernova, Li Zhou, Savannah E Jacobs, Duc Duong, Hiroaki Kiyokawa, Yury O Chernoff, Ming Li, Natalia Shcherbik, Bo Zhao, Jun Yin

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.

Comparing The Performance Of A Targeted Pull-Down Assay To Shotgun Sequencing For Improving Respiratory Infectious Disease Surveillance, Monica R. Christian

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Current surveillance focuses on well characterized pathogens such as influenza. Since 2000, there have been multiple outbreaks of respiratory disease. These outbreaks have demonstrated the need for robust and collaborative global efforts to identify, monitor, and contain novel respiratory viruses. This study aims to improve the ability of public health agencies to monitor and respond to respiratory disease outbreaks. Using five respiratory pathogens, this study compares a molecular capture technology from Twist Biosciences to the shotgun sequencing approach of whole transcriptome amplification (WTA, Qiagen) with the goal of determining which method is most effective using cost, usability, and sequencing quality …

Regulation Of Erk3 By Kras Signalling And Its Role In The Growth Of Lung Adenocarcinoma (Luad) Cells, Shreya Akunapuram

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Extracellular signal related kinase 3 (ERK3) is one of the atypical mitogen activated protein kinases (MAPK). It is expressed ubiquitously and plays a role in a variety of cellular processes, including cell growth and differentiation. ERK3’s role in promoting migration and invasion in various cancers has been well established. ERK3 is upregulated in non-small cell lung cancers (NSCLCs) and has been shown to promote NSCLC tumor growth and progression. However, the regulation of ERK3 in lung cancers remains largely unclear. A recent study indicates that ERK3 phosphorylation at S189, an indicator of ERK3 activity, is upregulated by KRAS in NSCLCs. …

The Effect Of Pomegranate Consumption On The Gut Microbiome, Brant Bandow

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Pomegranate is known to have antioxidant and prebiotic qualities that have been shown to promote the growth of beneficial bacteria while reducing inflammation in the gut. Inflammation in the gut is an issue that results in many health problems including obesity and colon cancer. In this study, an experimental group received a daily pomegranate supplement for three weeks where a control group did not receive any supplement. After sequencing gDNA isolated from fecal samples from both before and after the trial period there was a significant difference between the two groups. The largest amount of variability is attributed to the …

A Novel Role Of Lipin1 In The Regulation Of Expression And Function Of Nnos, Ayat Azzam

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Duchenne muscular dystrophy (DMD) is a severe and progressive muscular dystrophy that develops in the skeletal muscles because of mutations in the dystrophin gene. Dystrophin stabilizes sarcolemma and assembles neuronal nitric oxide synthase (nNOS) into the dystrophin-associated protein complex on the sarcolemma. The absence of dystrophin triggers the delocalization of nNOS and contributes to the misregulation of muscle development, blood flow, muscle fatigue, and inflammation. Lipin1 was reduced in the skeletal muscles of patients with DMD and the mdx mouse model of DMD. In this study, we explored the role of lipin1 in the regulation of nNOS expression and function. …

Molecular Investigation Of Minor Genomic Populations And Biological Exposures In Human Health, Brandon Ned Johnson

Dissertations and Theses

The study of genetics has contributed to countless discoveries related to human health and disease. However, the complexities of human biology reside not only in the genome but also in the contributions from environmental exposures, as measured via the classical twin design. To understand the influence of biological exposures, I implemented study designs to explore both the health associations and propagation of foreign genetic material. Microchimerism has been studied for association with several clinical conditions, and I further investigated if male microchimerism could elucidate the etiology of Mayer-Rokitansky-Küster-Hauser syndrome. Prevalence of male microchimerism in women with no history of pregnancy …

Dissecting Interactions Across Gene Regulatory Layers In C. Elegans, Morgan Taylor

Biological Sciences Theses and Dissertations

The nematode Caenorhabditis elegans is a powerful tool for studying nervous system genetics. Though relatively simple compared to mammals, C. elegans boasts a remarkably well-conserved neuronal genome and proteome, and its utility in the characterization of neuronal genes has been well-established. However, gene expression is often controlled by complex interactions between multiple genes, and teasing apart the functions of individual genes within such networks remains a challenge. Dissecting these interaction networks is crucial in determining the multifaceted functions of important, conserved regulatory genes. Here we explore interactions between gene regulatory layers in the C. elegans nervous system, employing a synthetic …

Elucidating Mechanisms Of Biofluorescence And Bioluminescence In Marine Organisms, Andrew M. Guarnaccia

Dissertations, Theses, and Capstone Projects

Biofluorescence and bioluminescence are two methods of light emission that entail separate mechanisms of action but end at the same process: a colorful display that have tremendous ecological and behavioral benefits, whether it be used to communicate with conspecifics, camouflage into a multicolored background, attract unsuspecting prey, or alert others to a predator. In biofluorescence, higher-energy, shorter wavelength light is absorbed then re-emitted as lower-energy, longer-wavelength light. Bioluminescence on the other hand entails a chemical reaction in which a small molecule is oxidized by an enzyme, creating a high-energy intermediate that sheds the excess energy in the form of visible …

Assembly Of The Peripheral Arm Subunits Of Escherichia Coli Complex I And Analysis Of Clinical Mutations, Hind Alkhaldi

Biological Sciences Theses and Dissertations

Respiratory Complex I from E. coli is a proto-type of the mitochondrial enzyme, consisting of a 6-subunit peripheral arm (B-CD-E-F-G-I) and a 7-subunit membrane arm. When subunits E-F-G (N-module), were expressed alone they formed an active complex as determined by co-immunoprecipitation and native gel electrophoresis. When co-expressed with subunits B and CD, only a complex of E-F-G was found. When these five subunits were co-expressed with subunit I and two membrane subunits, A and H, a complex of B-CD-E-F-G-I was membrane-bound, constituting the N- and Q-modules. Assembly of Complex I was also followed by splitting the genes between two plasmids, …

Development And Analysis Of Next-Generation Polymeric And Bio-Ceramic Based Orthopedic Scaffolds By Advanced Manufacturing Techniques, Sudeep K. Gummadi

ETD Archive

Gliomas express mutant isocitrate dehydrogenases producing excessive amounts of D 2-hydroxyglutarate (D2HG) and releasing some of it into the environment. The immune surveillance is reduced as a result, however, the mechanisms behind lymphocyte suppression by the D2HG stereoisomer remain unknown. I incubated Jurkat T cells with D2HG at concentrations present within and surrounding gliomas, or its obverse L2HG stereoisomer, and quantified 2HG isomers within washed cells by TSPC derivatization with stable isotope-labeled D2HG and L2HG internal standards, HPLC separation, and mass spectrometry. D2HG was found in quiescent cells in double the amount of L2HG. External D2HG or L2HG increased the …

Multicopy Suppression Of Ribosomal Protein Deletion Mutants, Jacqualine Cerbone

Senior Honors Projects

To suppress the ribosome assembly defective phenotype of a ribosomal protein uS17- deficient mutant, various ribosomal proteins were used to act as multicopy suppressors. Ribosomal protein uS17 is a highly conserved component of the 30S (small) ribosomal subunit and is important for 30S subunit assembly. Mutants with a deletion of rpsQ (the gene encoding uS17) are viable but have a severe 30S subunit assembly defect and a temperature-sensitive (ts) phenotype. While fully assembled 30S subunits do form, incomplete ("20S") particles lacking several proteins accumulate. We hypothesized that increasing the intracellular concentration of one or more of the proteins missing from …

Genomic Instability At A Polypurine/Polypyrimidine Repeat Sequence, Nathen S. Zavada

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Microsatellite repeat sequences have been shown to induce replication stalling, fork collapse, double-strand breaks (DSBs), and possibly stimulate break-induced replication. In this study we use a dual-fluorescent HeLa model that is designed to monitor recombination at an ectopic site through use of flow cytometry and inverse PCR with a microsatellite in the lagging strand for DNA synthesis. To test the stability of the 78 bp polypurine/pyrimidine repeat from the PDK1 locus, we subjected cells to replication stress drugs designed to induce DSBs and measure break-induced replication (BIR). The study revealed that polypurine repeat cells undergo endogenous stress contributing to instability …

Instability And Extrachromosomal Circular Dna Formation At Microsatellites And Unstable Dna Sequences, Matilyn M. Shanahan

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We have previously documented our evidence of genetic instabilities at the (Pu/Py)78 and (ATTCT)47 sequences and our reasoning for identifying break-induced replication (BIR) as the mode of repair responsible for the mutations in the DNA flanking the unstable inserts. Now, as our lab investigates the protein mechanisms at play in the BIR pathway taking place at these sites, we are also expanding our knowledge of how this mechanism extends into the pathways responsible for forming extrachromosomal circular DNA (eccDNA) molecules. We have documented the phenomena posed as the driving factors for eccDNA formation in our systems containing (Pu/Py)78 and (ATTCT)47. …

Effects Of Lipin1 Deficiency & Restoration In The Dystrophic Diaphragm, Alexandra Brown

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Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder that is characterized by severe and progressive muscle wasting (Venugopal & Pavlakis, 2021). This disease is caused by a mutation in the largest known human gene which encodes the protein, dystrophin (Gao & McNally, 2015). Dystrophin connects the inner cytoskeleton to the extracellular matrix and is critical for maintaining the structural stability of muscle cells during contraction (Venugopal & Pavlakis, 2021). Mutations to the dystrophin gene result in myocyte membrane instability, contributing to the structural deterioration of the muscle tissue (Venugopal & Pavlakis, 2021). Progressive muscle degeneration and the replacement of …

The Role Of Pi4kb In Cellular Localization Of Small Gtpases, Parisa Sadrpour

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Constitutively active oncogenic mutant K-Ras is one the principal contributors to human cancers including 90% of pancreatic, 50% of colorectal and 32% of non-small cell lung cancers. However, except for K-Ras G12C oncogenic mutant, which only presents in about 13% of non-small cell lung cancer patients, there is no anti-K-Ras therapy for a considerable subset of K-Ras mutations in human tumors, reflecting challenges for targeting oncogenic K-Ras activity. K-Ras is a membrane-bound small GTPase; when active, it triggers multiple signaling pathways regulating a variety of key cellular functions such as cell growth, proliferation and survival. To initiate these signaling cascades, …

Reconstruction Of Gut Microbiome Via Intermittent Feeding, Kourtney Sprague

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The benefits of intermittent fasting have been studied across many facets of health. It is known that physiologically fasting results in a metabolic switch from liver-derived glucose to adipose cell derived ketones to be used as energy and signaling molecules. Fasting down-regulates inflammation, increases expression of antioxidant defenses, and activates pathways for DNA repair and autophagy. While fasting or intermittent feeding effects on host physiology have been identified, the relationship between fasting and gut microbiome is not as well known. In times of gut rest, resident microbes undergo detoxication and motility, and in times of activity, gut microbes undergo DNA …

Molecular Mechanisms Of Aberrant Protein Glycosylation In Pancreatic Cancer Stemness And Metastasis, Frank Leon

Theses & Dissertations

A myriad of genetic and other abnormal changes underlies the aggressiveness and dissemination properties observed in pancreatic cancer (PC). Aberrant protein glycosylation is a commonly observed feature in PC. The modification of protein O-glycosylation is mediated by glycosyltransferases, which attach and sequentially elongate monosaccharides on Serine/Threonine (Ser/Thr) motifs. Aberrant glycosylation is recognized as an emerging hallmark of cancer where a disruption in normal glycosylation results in irregular O-glycans.

This dissertation research has investigated the consequences of aberrant protein glycosylation on stemness and enhancement of metastatic properties in pancreatic ductal adenocarcinoma (PDAC). Several publications have reported aberrant O-glycosylation increases in oncogenic …

Purification And Functional Characterization Of The Iron-Responsive Transcription Factor Aft1 From C. Glabrata, Jade Ikahihifo-Bender

Senior Theses

Due to its unique ability to serve as both an electron donor and acceptor, iron is utilized as a co-factor for many biological processes, including electron transfer, oxygen binding, and vitamin synthesis. Iron is also a key factor during fungal infections as the human host and invading pathogens battle over limited iron pools. The primary iron-responsive transcription factor Aft1 in the opportunistic pathogenic yeast Candida glabrata responds to iron deficiency by activating expression of iron acquisition genes. However, the mechanisms for sensing intracellular iron levels and regulating Aft1 activity in response to iron are unknown. The C. glabrata iron regulation …

Potential Drug Treatment For Duchenne Muscular Dystrophy Which Could Be Through Upregulation Of Lipin1, Rajsi Y. Thaker

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Duchenne muscular dystrophy (DMD) is a genetic disorder leading to progressive muscle degeneration and weakness due to mutation in dystrophin gene, which is very important for maintaining muscle membrane integrity. Dystrophin is the largest gene in the human genome therefore more prone to mutation. There is currently no cure for DMD. Our lab recently found that Lipin1 deficient myofibers showed upregulation of necroptosis correlated with the loss of muscle membrane integrity. Our primary approach for ameliorating dystrophic phenotype in DMD is through reduction of necroptosis using drugs which can potentially upregulate Lipin1 expression. In this study, we identified two drugs …

Quantitated Effects Of Nutritional Supplementation On Exercise Induced Sweat, Andrew Blake Austin Browder

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Discovery studies have identified many metabolites contained in human sweat. However, quantitative analysis of the sweat metabolome content remains mostly unknown. Furthermore several attributes, including rate, have been defined to affect sweat metabolite content, while other effectors, like diet, remain unknown. This study works to quantitatively define the metabolite impact caused by nutritional supplementation. To better understand the effect diet plays, a LC-MS method was developed focusing on improving resolution and peak width. While the literature provided examples of how diet affected sweat metabolite concentrations, the long-term effects of diet have not been explored. The experiment described here attempts to …

Let-Dependent Low Dose And Synergistic Inhibition Of Human Angiogenesis By Charged Particles: Validation Of Mirnas That Drive Inhibition, Amber M. Paul, Yen-Ruh Wuu, Burong Hu, Hazeem Okunola, Elizabeth A. Blaber, Margareth Cheng-Campbell, Afshin Beheshti, Peter Grabham

Publications

Space radiation inhibits angiogenesis by two mechanisms depending on the linear energy transfer (LET). Using human 3D micro-vessel models, blockage of the early motile stage of angiogenesis was determined to occur after exposure to low LET ions (/AMU), whereas inhibition of the later stages occurs after exposure to high LET ions (>8 KeV/AMU). Strikingly, the combined effect is synergistic, detectible as low as 0.06 Gy making mixed ion space radiation more potent. Candidates for bystander transmission are microRNAs (miRNAs), and analysis on miRNA-seq data from irradiated mice shows that angiogenesis would in theory be downregulated. Further analysis of three …