Biochemistry Commons^™

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

Theses and Dissertations

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

Mechanisms By Which Mnte-2-Pyp Suppresses Prostate Cancer Cell Growth, Yuxiang Zhu

Theses & Dissertations

Prostate cancer patients are often treated with radiotherapy. MnTE-2-PyP, is a superoxide dismutase (SOD) mimic and a known radioprotector of normal tissues. Our recent work demonstrates that MnTE-2-PyP also inhibits prostate cancer progression with radiotherapy; however, the mechanisms remain unclear. In this thesis, we identified that MnTE-2-PyP-induced intracellular H2O2 levels are critical in inhibiting growth of prostate cancer cells. We found that MnTE-2-PyP induced protein oxidations in PC3 cells and one major group of oxidized protein targets were involved in energy metabolism. The oxidative phosphorylation rates were significantly enhanced in both PC3 and LNCaP cells with MnTE-2-PyP treatment, but mitochondrial …

Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma

Doctoral Dissertations

The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while protecting internal tissues and maintaining symbiotic relationship with luminal microbiota. This bio-barrier consists of a cellular epithelial barrier and an acellular mucus barrier. Secreted mucus regulates barrier function via in situ biochemical and biophysical interaction with luminal content that continually evolves during digestion and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain homeostasis in the gastrointestinal tract. However, the importance of mucus barrier is largely underrated for in vitro mucosal tissue modeling. The major gap is the lack of experimental material …

Sine Oculis Homeobox Homolog 1 (Six1) Plays A Critical Role In The Progression Of Pulmonary Fibrosis., Cory Wilson

Dissertations & Theses (Open Access)

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia with a median survival time of 2-4 years after diagnosis. The alarming mortality rate is due to the lack of effective treatments. IPF is a chronic disease that is characterized by alveolar destruction due to increasing extracellular matrix deposition that leads to poor lung compliance, impaired gas exchange, and ultimately respiratory failure. Repetitive alveolar epithelial injury is a central process to the underlying pathology with injury to the type II alveolar epithelial cells (AT2) specifically being a key player in the pathogenesis of IPF. Recent studies have shown that …

Ionic Mechanism Of Lysosomal Function And Cell Metabolism, Jian Xiong

Dissertations & Theses (Open Access)

Two Pore Channels (TPCs) are endolysosomal ion channels that are permeable to sodium and calcium. Defects in TPCs have been implicated to impair vesicle trafficking, autophagy and cell metabolism control; however, the detailed mechanism remains largely unknown. In this study, I show that TPCs are critical for appropriate cargo delivery to the lysosomes and deletion of either TPC1 or TPC2 leads to delayed clearance of autophagosomes, resulting in enlarged lysosomes and accumulated contents inside the lysosomes. Cells with both TPC deleted also exhibit 50% reduction in lysosomal amino acids under normal culture conditions, leading to reduced homeostatic mTORC1 activation.

Glutamine …

Dnajc7, A Molecular Chaperone Protein That Modulates Protein Misfolding In Amyotrophic Lateral Sclerosis (Als), Meaghan Kathleen Stoltz

Electronic Thesis and Dissertation Repository

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease associated with protein misfolding and dysregulated cellular protein quality control mechanisms. Molecular chaperones, and heat shock proteins (Hsp), are key players in maintaining cellular protein quality control. DNAJC7 is an understudied cytosolic Hsp40 that works together with Hsp70 and Hsp90 to regulate proper protein folding or degradation. Of note, mutations in the gene encoding DNAJC7 were discovered to cause familial ALS. We asked whether ALS-associated mutations in DNAJC7 compromise its function as a chaperone, which may cause the toxic accumulation of misfolded proteins. This study attempts to uncover the functions of DNAJC7 …

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

Dissertations & Theses (Open Access)

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

Investigating Chitosan Modified With Triethylammonium Butanamide And Triethylphosphonium Butanamide As Non-Viral Gene Delivery Vectors By Examining Cytotoxicity And Transfection Efficiency, Deborah C. Ehie

MSU Graduate Theses

Gene therapy is a very challenging field, especially with new emerging genetic disorders. Chitosan (CS), due to chitosan’s flexibility, biocompatibility, and biodegradability, has been of interest in the world of gene therapy especially as researchers are gravitating towards non-viral vectors due to the problems caused by viral vectors. Nevertheless, there are still issues regarding solubility, cellular uptake of cargos being transported in vitro or in vivo, increased cytotoxicity levels, as well as many other things that prevent chitosan from being an efficient gene delivery agent. Here I present five derivatives of chitosan, which were all modified with either triethylphosphonium …

Insights Into Desiccation Tolerance: Properties Of Late Embryogenesis Abundant Proteins From Embryos Of Artemia Franciscana, Blase Matthew Leblanc

LSU Doctoral Dissertations

LEA proteins are a family of intrinsically disordered proteins that are expressed in various life stages of anhydrobiotic organisms and have been strongly associated with survival during water stress. The brine shrimp Artemia franciscana is the only known anhydrobiotic animal that expresses LEA proteins from Groups 1, 3, and 6. Here, I report that AfrLEA6, a novel Group 6 LEA protein, is most highly expressed in embryos during diapause and decreases throughout pre-emergence development. Notably, there is an acute drop in expression upon termination of the diapause state and the titer of AfrLEA6 during diapause is 10-fold lower than values …

Development Of Ligand Guided Selection (Ligs) To Identify Specific Dna Aptamers Against Cell Surface Proteins, Hasan Ekrem Zumrut

Dissertations, Theses, and Capstone Projects

Oligonucleotide aptamers (nucleic acid-based affinity reagents) are an emerging class of synthetic molecules that display high affinity and specificity towards their targets. Aptamer molecules for a target of interest are obtained using a combinatorial chemistry-based method termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX is an in vitro selection process in which a random oligonucleotide library is subjected to repeated cycles of target incubation, separation, and amplification until target-specific evolved sequences become prevalent in the library. Typically, SELEX is used against target molecules such as small molecules and proteins, in their purified state. However, aptamers selected against purified …

Post-Lysosomal Cholesterol Trafficking: Novel Tools And Insights, Mckenna Rae Feltes

Arts & Sciences Electronic Theses and Dissertations

Cholesterol is an essential mammalian lipid. It is a major component of cellular membranes, a precursor molecule for the synthesis of hormones and bile acids, and a regulator of protein function. Although cholesterol is synthesized, de novo, in the endoplasmic reticulum, cells principally meet cholesterol requirements through uptake of lipoprotein particles. Lipoprotein-derived cholesterol is transported to the lysosome where it is transferred from the soluble lysosomal protein, NPC2, to limiting-lysosomal membrane protein NPC1. Cholesterol is then re-distributed to other cellular membranes in order to fulfill organellar cholesterol requirements; however, the cellular machineries involved in coordinating this distribution are poorly characterized. …

Breast Cancer Sub-Clones That Metastasize To Lung And Bone Exhibit Different Metabolic Preferences, Mollie Merrell

Honors Theses

Metastasis is responsible for the majority of cancer related deaths. In breast cancer the lungs and bones are the major sites for metastasis. Previous studies used the metastatic aggressive MDA-MB-231 breast cancer line to isolate sub-clones that preferentially invade the lungs (LM line) or bones (BoM line). While genes associated with the tissue specific metastasis have been identified, it is unknown if metabolic adaptations contribute to the growth of the LM and BoM lines in their respective organs. The goal of this study was to test the hypothesis that the LM and BoM lines exhibit differences in glucose and glutamine …

Molecular Insights Into Paf-1 Mediated Pancreatic Homeostasis, Stemness, And Cancer Progression, Saswati Karmakar

Theses & Dissertations

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that has one of the lowest 5-year survival rates among cancers, at just 9%. This grim prognosis is primarily due to the extensive metastatic spread of tumor cells beyond the pancreas at diagnosis and the inability of current therapeutic modalities to treat this aggressive disease effectively. Given that the cancer cells in pancreatic tumors are heterogeneous, the major culprit for cancer initiation, progression, and metastasis remains elusive. Recent studies provide evidence for the existence of highly tumorigenic and drug-resistant cells that are capable of tumor initiation, known as the cancer stem cells …

Glucose Metabolism Of Breast Cancer Sub-Clones That Preferentially Metastasize To The Lungs And Bone, Anna G. Skubiz

Honors Theses

Malignant breast cancers exhibit preferential metastasis to bone and lung (1). While changes in gene expression in lung-specific (LM) and bone-specific metastasis (BoM) lines derived from the MDA-MB-231 breast cancer line have been identified, few metabolic genes are differentially expressed; thus it is unknown if tissue-specific metabolic reprogramming occurs. Two hallmarks of cancer cells are an altered metabolic phenotype characterized by enhanced conversion of glucose to lactate in spite of adequate oxygen availability for complete mitochondrial oxidation of this substrate (referred to as aerobic glycolysis or the Warburg effect) and a greater dependence on glutamine. These changes in primary tumor …

Cell Proliferation And Viability Inhibition By Resveratrol On Breast Cancer Cell Lines, Kyle Ford Gordon Jr

Honors Theses

Antioxidants are well-known for their various health benefits. They are able to protect cells from being damaged by free radicals that are produced by vital biochemical processes. It has long been known that antioxidants are important in our everyday health, but their potential as disease preventers and potential therapeutic agents is a relatively new field of study. Resveratrol, a natural polyphenol and well-known antioxidant, is found in plants, fruits, and products derived from them, like red wine. Resveratrol has been shown to have various properties, including antiaging, anti-aggregation of platelets, anti-inflammatory, and anticancer activities. Because of their many health benefits, …

Investigating The Redox Sensitivity Of Mitf Splice Variants, Rachel Berryman

Senior Theses

Within pigment-producing cells known as melanocytes, the transcription factor MITF is intimately involved in regulating genes associated with cell cycle maintenance and melanocyte differentiation. Research, however, has provided conflicting results on the relationship between the expression levels of MITF and melanocyte cell fate. To complicate matters, two splice variants of MITF exist, differing by only 18 base pairs. These variants have been observed at variable levels of expression in melanocyte and melanoma cells, raising the question as to their functional purpose. Building upon previous research by the Leachman/Cassidy lab that identified the redox sensitivity of MITF while additionally establishing a …

Cloning, Expression, Purification And Characterization Of Heparin-Binding Pocket Of Recombinant Fgf1, Quratulayn Ashraf

Graduate Theses and Dissertations

Fibroblast growth factors are polypeptide members of the FGF family, which to date comprises of at least 22 members. They belong to a group of growth factors and are involved in a variety of cellular processes including wound healing, angiogenesis, differentiation and development (organogenesis). Amongst FGF members, human acidic FGF-1 and basic FGF-2 are the most characterized. FGF-1 and FGF-2 are known to share more than 80% sequence similarity and have an identical structural fold. However, their biological roles are quite different. FGFs bind to heparin and heparan sulfate ligands through their heparin-binding pockets. The interactions are primarily electrostatic in …

Tpr-Containing Proteins Control Protein Organization And Homeostasis For The Endoplasmic Reticulum, Jill Bradley-Graham

Doctoral Dissertations

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into several functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Nine TPR-containing proteins have been shown to localize to the ER and control protein organization and …

The Role Of Apkcs And Apkc Inhibitors In Cell Proliferation And Invasion In Breast And Ovarian Cancer, Tracess B. Smalley

USF Tampa Graduate Theses and Dissertations

Research has demonstrated that the atypical protein kinase C-zeta (PKC-ζ) is a component of many dysregulated pathways in breast and ovarian cancer, including cellular proliferation, survival, and cell cycle upregulation. Breast and ovarian cancers affect women every day and are second and fifth leading cause of cancer death. Women who seek treatments are commonly met with invasive surgeries or chemotherapy. Protein kinase C (PKC) is a family of serine and threonine phosphorylating kinases that have been shown to modulate and transduce signaling cascades that play roles in the development and survival of cancers. Atypical PKC (aPKC), have been heavily suggested …

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 …

Thyroxine-Dependent And -Independent Effects On Premature Aging And Myelination In Atrx Mutant Mice, Megan E. Rowland

Electronic Thesis and Dissertation Repository

ATRX is an ATP-dependent chromatin remodeler required to safeguard genomic integrity. Conditional deletion of Atrx in the mouse embryonic forebrain and anterior pituitary in Atrx^Foxg1Cre mice phenocopies mouse models of progeria which display increased DNA damage, coupled with reduced lifespan, growth and subcutaneous fat. These mice also have severely low circulating levels of insulin like growth factor 1 (IGF-1) and (T4) which have been reported in models of premature aging. Based on evidence that Igf1 is activated by the ligand-bound thyroid hormone receptor, I tested whether T4 supplementation could restore IGF-1 levels and ameliorate premature aging phenotypes in Atrx …

Through The Back Door: Proteins Escape Cells Without Conventional Permission, Michael J. Cohen

Dissertations, Theses, and Capstone Projects

Proteins secreted to the extracellular environment play a fundamental role as signals, in metabolism, and a variety of other processes. The process of secretion through the endoplasmic reticulum and Golgi to the plasma membrane is well documented, and all cargo in this pathway contains a signal peptide. However, a variety of proteins secreted from eukaryotes lack a signal peptide and are called unconventionally secreted proteins. Here we discuss known mechanisms of unconventional protein secretion, as well as model proteins which follow characterized pathways. Additionally, we summarize the roles various unconventionally secreted proteins play outside of cells and suggest criteria for …

Quantification Of Mitochondrial Zinc Homeostasis And Analysis Of Zinc And Polyamine-Mediated Axonal Trafficking, Dylan Hunt Fudge

Electronic Theses and Dissertations

The zinc ion is an important emerging signaling molecule for biological processes. In this work we engineered improved zinc sensors based on our previously developed fluorescent sensor GZnP1 to provide sensors with a higher fluorescent readout, faster kinetics, and a superior sensitivity to zinc. We utilized these zinc sensors and further developed the sensors to answer questions pertaining to biological zinc. We showed the labile zinc concentration in the mitochondrial matrix was less than 1 pM while the labile zinc concentration in the cytosol and mitochondrial IMS were comparable (~100 pM) across four different cell lines. Using these zinc sensors …

Sorting Of Cargo Proteins Within The Regulated Secretory Pathway: The Peripheral Membrane Protein Hid-1 As A Sorting And Vesicle Biogenesis Factor, Blake H. Hummer

Electronic Theses and Dissertations

Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize …

Variations On A Theme: Intricacies Of Unanchored Poly-Ubiquitin Signaling And Toxicity, Jessica Renee Blount-Pacheco

Wayne State University Dissertations

Ubiquitin is an 8.5 kDa post-translational modifier involved in essentially all eukaryotic cellular processes. Through a process called ubiquitination, ubiquitinating enzymes chemically attach ubiquitin to substrate proteins to control their fates, resulting in anything from their recruitment into signaling pathways to their proteasomal degradation, with a plethora of possibilities in between. Ubiquitin molecules can also be attached to one another, resulting in poly-ubiquitin chains with various effects depending on the number of ubiquitin molecules and the specific amino acid residues used to link them together. While most poly-ubiquitin in the cell exists as conjugated species, there are also untethered poly-ubiquitin …

Therapeutic Dna: Delivery And As A Delivery Vehicle, Natalie J. Holl

Masters Theses

"A review of gene delivery methods and gene editing methods, as well as original research utilizing DNA as a delivery vehicle is presented in the following thesis. Thousands of diseases have been linked to genes. Gene therapy, either delivering therapeutic genes or editing DNA bases, has arisen as a treatment option with the potential to cure diseases, rather than just ease symptoms. Genes and editing tools need to be delivered to cells for these therapies to be effective and many techniques have been developed to address the issue of delivery. Nonviral and viral methods have been used to deliver nucleic …

Characterization Of The Influences Of Human Cytomegalovirus Glycoprotein O (Go) Expression On Gh/Gl Complexes Assembly And Its Polymorphisms On Cell-Free And Cell-To-Cell Spread, And Antibody Neutralization., Le Z. Day

Graduate Student Theses, Dissertations, & Professional Papers

Human cytomegalovirus (HCMV) is widely spread throughout the world and immunocompromised individuals can suffer severe diseases from HCMV infection. Once the infection is established, HCMV can spread through the body and infect many major somatic cell types. The glycoproteins H and L (gH/gL) on HCMV envelope can be bound by either gO or the UL128-131 proteins to form complexes gH/gL/gO and gH/gL/UL128-131 that are critical for viral entry and spread, and these two complexes are important targets of neutralizing antibodies. Strains of HCMV vary considerably in the levels of gH/gL/gO and gH/gL/UL128-131. gO is one of the most diverse loci …

Seasonal Variation In Mitochondrial Bioenergetics Of The Bluegill Sunfish, Lepomis Macrochirus, From A Shallow Midwest River, Derick Isaac Lamptey

Masters Theses

As average global temperature increase, the frequency and magnitude of extreme temperatures in shallow aquatic ecosystems are more ubiquitous. In order to understand how these changing thermal regimes affect aquatic ectotherms, it is essential to develop studies evaluating the response of ectotherms to seasonal fluctuating thermal regimes. Previous studies on fluctuating temperature regimes have reported an increased physiological stress leading to morphological, behavioral and biochemical adaptations. From the latter, the adaptive capacity and seasonal performance associated with optimal function of the oxidative phosphorylation system (OXPHOS) are key for species persistence. However, studies on this matter are scarce. This study explores …

Investigation Of Glutamate Carboxypeptidase Ii Roles In The Folate Cycle Dependent Reproduction And Development Of Caenorhabditis Elegans, Jessica M. Derham

Masters Theses

Glutamate Carboxypeptidase II (GCPII) is a transmembrane, zinc metallopeptidase that is expressed in a wide range of organisms, including roundworms, mice, and humans. In humans, GCPII is primarily expressed in the prostate, kidneys, small intestine, and central nervous system. Within the small intestine, the expected function of GCPII is to aid in the absorption of dietary folate from the intestinal lumen. GCPII cleaves excess glutamates from folate to yield monoglutamated folate which is then readily transported into the enterocyte. Folate can then be used through the one carbon metabolic cycle for the synthesis of nucleotides, conversion of homocysteine to methionine, …

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

Graduate Theses, Dissertations, and Problem Reports

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