Biochemistry, Biophysics, and Structural Biology Commons^™

The Non-Canonical Hydroxylase Structure Of Yfcm Reveals A Metal Ion-Coordination Motif Required For Ef-P Hydroxylation, Kan Kobayashi, Assaf Katz, Andrei Rajkovic, Ryohei Ishii, Owen E. Branson, Michael A. Freitas, Ryuichiro Ishitani, Michael Ibba, Osamu Nureki

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a β-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The β-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any …

Large-Scale Identification Of Chemically Induced Mutations In Drosophila Melanogaster, Nele A Haelterman, Lichun Jiang, Yumei Li, Vafa Bayat, Hector Sandoval, Berrak Ugur, Kai Li Tan, Ke Zhang, Danqing Bei, Bo Xiong, Wu-Lin Charng, Theodore Busby, Adeel Jawaid, Gabriela David, Manish Jaiswal, Koen J T Venken, Shinya Yamamoto, Rui Chen, Hugo J Bellen

Faculty, Staff and Students Publications

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of …

Identification And Characterization Of A Functional L-Gulono-1,4-Lactone Oxidase In Arabidopsis Thaliana, Siddique Aboobucker

Student Theses and Dissertations

Vitamin C (ascorbate, AsA) is essential for plant and animal health. We cannot synthesize AsA on our own and therefore we must consume it from fruits and vegetables. Ascorbate plays multiple roles in plant physiology, and increasing AsA content in plants remains challenging. There are four biosynthetic AsA pathways in plants: the D-mannose/L-galactose, L-gulose, D-galacturonate, and myo-inositol routes. The AsA metabolic network is not completely understood. The inositol pathway has four enzyme-catalyzed steps, and our group has made significant progress on characterizing the first three enzymes. This work focuses on L-gulono-1,4-lactone oxidase (GulLO), the last enzyme in the inositol pathway …

Mechanisms And Molecular Biology Of Major Tumor Suppressors, Brienne E. Engel

USF Tampa Graduate Theses and Dissertations

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively.

RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes …

Role And Regulation Of Snon/Skil And Plscr1 Located At 3q26.2 And 3q23, Respectively, In Ovarian Cancer Pathophysiology, Madhav Karthik Kodigepalli

USF Tampa Graduate Theses and Dissertations

Ovarian cancer is one of the most common causes of gynecological cancer related deaths in women. In 2014, the estimated number of deaths due to ovarian cancer is 14,270 with occurrence of over 22, 240 new cases (National Cancer Institute, http://seer.cancer.gov/statfacts/html/ovary.html). Despite improvement in treatment strategies, the 5-year survival rate is still below 50% mainly due to chemoresistance and relapse. Amplification of chromosomal region 3q26 is a common characteristic in various epithelial cancers including ovarian cancer. This region harbors various oncogenes including the TGFβ signaling mediators EVI1 and SnoN/SkiL, PKCι and PIK3CA amplified at 3q26.2 and 3q26.3, respectively, in ovarian …

Eukaryotic Gene Expression Patterns Of Microorganisms In The Amazon River Plume Parallel The Biogeochemistry Of Plume Waters, Brian L. Zielinski

USF Tampa Graduate Theses and Dissertations

Unraveling the microbiological processes that occur as water travels from a river's mouth into the ocean is critical to understanding the role of river plumes in global biogeochemical cycles. Metranscriptomics, the gene expression of a whole community of organisms, was utilized to examine six stations along the Amazon River Plume (ARP) in 2010 to test thehypothesis that there were measurable differences in gene expression for key biogeochemical genes along the ARP. This body of work focuses on methods developed to identify which genes are biogeochemically important for a particular environment along extreme salinity, nutrient and community gradients in the ARP, …

Mistranslation Of The Genetic Code, Adil Moghal, Kyle Mohler, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

During mRNA decoding at the ribosome, deviations from stringent codon identity, or “mistranslation,” are generally deleterious and infrequent. Observations of organisms that decode some codons ambiguously, and the discovery of a compensatory increase in mistranslation frequency to combat environmental stress have changed the way we view “errors” in decoding. Modern tools for the study of the frequency and phenotypic effects of mistranslation can provide quantitative and sensitive measurements of decoding errors that were previously inaccessible. Mistranslation with non‐protein amino acids, in particular, is an enticing prospect for new drug therapies and the study of molecular evolution.

Relaxed Substrate Specificity Leads To Extensive Trna Mischarging By Streptococcus Pneumoniae Class I And Class Ii Aminoacyl-Trna Synthetases, Jennifer Shepherd, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances by cis- and trans-editing pathways, it has been revealed that in organisms such as Streptococcus pneumoniae some aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNA^Ile with both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent …

Role Of Vitamin D3 In Modulation Of Δnp63Α Expression During Uvb Induced Tumor Formation In Skh-1 Mice, Natasha Tremayne Hill, Gabriel H. Gracia-Maldondo, Mary K. Leonard, Amanda R. Harper, Kathleen L. Tober, Tatiana M. Oberyszyn, Madhavi P. Kadakia

Biochemistry and Molecular Biology Faculty Publications

ΔNp63α, a proto-oncogene, is up-regulated in non-melanoma skin cancers and directly regulates the expression of both Vitamin D receptor (VDR) and phosphatase and tensin homologue deleted on chromosome ten (PTEN). Since ΔNp63α has been shown to inhibit cell invasion via regulation of VDR, we wanted to determine whether dietary Vitamin D₃ protected against UVB induced tumor formation in SKH-1 mice, a model for squamous cell carcinoma development. We examined whether there was a correlation between dietary Vitamin D₃ and ΔNp63α, VDR or PTEN expression in vivo in SKH-1 mice chronically exposed to UVB radiation and fed chow containing …

Activation Of Dna Damage Checkpoint Pathways During Skeletal Myoblast Differentiation And Apoptosis, Mofetoluwa Oluwasanmi, Greg Kliment, Crystal M. Weyman

Undergraduate Research Posters 2014

A subset of skeletal myoblasts undergo apoptosis rather than differentiation when cultured in differentiation media (DM: absence of growth factors). While the muscle regulatory transcription factor MyoD is known to control the process of differentiation, our lab has recently discovered that MyoD is also controlling the apoptotic process in response to culture in DM by direct up-regulation of the pro-apoptotic Bcl2 family member PUMA. We similarly discovered that MyoD plays a role in the increased expression of PUMA and apoptosis in response to the DNA damaging agent, etoposide. This led to the hypothesis that culture in DM may lead to …

Nack Is An Integral Component Of The Notch Transcriptional Activation Complex And Is Critical For Development And Tumorigenesis, Kelly L Weaver, Marie-Clotilde Alves-Guerra, Ke Jin, Zhiqiang Wang, Xiaoqing Han, Prathibha Ranganathan, Xiaoxia Zhu, Thiago Dasilva, Wei Liu, Francesca Ratti, Renee M Demarest, Cristos Tzimas, Meghan Rice, Rodrigo Vasquez-Del Carpio, Nadia Dahmane, David J Robbins, Anthony J Capobianco

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are …

Investigating Propargyl-Linked Antifolates In Inhibiting Bacterial And Fungal Dihydrofolate Reductase, Joshua Andrade

Honors Scholar Theses

Antimicrobial agents have been invaluable in reducing illness and death associated with bacterial infection. However, over time, bacteria have evolved resistance to all major drug classes as a result of selective pressure. The advancement of new drug compounds is therefore vital. The Anderson-Wright Lab has focused on developing potent and selective inhibitors of dihydrofolate reductase (DHFR), an enzyme key in cell proliferation and survival, in several pathogenic species. The lab has found that a set of compounds, known as propargyl-linked antifolates, are DHFR inhibitors that are both biologically effective and have strong pharmacokinetic properties.

The efficacy of novel propargyl-linked antifolates …

Transcriptional Regulation Of Sinorhizobium Meliloti Cell Cycle-Related Genes In The Δcbra Mutant And Root Nodules Of Medicago Sativa, Corey S. Hazekamp

Graduate Masters Theses

Sinorhizobium meliloti is a Gram-negative alphaproteobacterium and nitrogen-fixing symbiont, which undergoes a novel cell cycle modification during its' host-microbe interaction. I intend to monitor the transcriptional regulation of cell cycle-related genes during free-loving growth, in addition to monitoring their expression during symbiosis. Using genes known to be regulated by CtrA in C. crescentus or predicted to be regulated by CtrA in S. meliloti, I aim to show how certain cell cycle genes are regulated in S. meliloti. In C. crescentus, CtrA acts as a transcription factor that is active when phosphorylated and inactive when not phosphorylated. In …

Translation Initiation Rate Determines The Impact Of Ribosome Stalling On Bacterial Protein Synthesis, Steven J. Hersch, Sara Elgamal, Assaf Katz, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Ribosome stalling during translation can be caused by a number of characterized mechanisms. However, the impact of elongation stalls on protein levels is variable, and the reasons for this are often unclear. To investigate this relationship, we examined the bacterial translation elongation factor P (EF-P), which plays a critical role in rescuing ribosomes stalled at specific amino acid sequences including polyproline motifs. In previous proteomic analyses of both Salmonella and Escherichia coli efp mutants, it was evident that not all proteins containing a polyproline motif were dependent on EF-P for efficient expression in vivo . The α- and β-subunits of …

Thiosulfoxide (Sulfane) Sulfur: New Chemistry And New Regulatory Roles In Biology, John Toohey, Arthur J L Cooper

NYMC Faculty Publications

The understanding of sulfur bonding is undergoing change. Old theories on hypervalency of sulfur and the nature of the chalcogen-chalcogen bond are now questioned. At the same time, there is a rapidly expanding literature on the effects of sulfur in regulating biological systems. The two fields are inter-related because the new understanding of the thiosulfoxide bond helps to explain the newfound roles of sulfur in biology. This review examines the nature of thiosulfoxide (sulfane, S0) sulfur, the history of its regulatory role, its generation in biological systems, and its functions in cells. The functions include synthesis of cofactors (molybdenum cofactor, …

Ef-P Dependent Pauses Integrate Proximal And Distal Signals During Translation, Sara Elgamal, Assaf Katz, Steven J. Hersch, David Newsom, Peter White, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the Δefp strain, with up to a 45-fold increase in ribosome density observed at …

Poly(Arginine) Derived Cancer-Targeting Peptides For The Development Of A Cancer-Targeted Gene Therapy Approach In Hepg2 Liver Cancer Cells, Stesha C. Joseph

Seton Hall University Dissertations and Theses (ETDs)

Cancer is a disease that has eluded medicinal approaches for many years and as a result new and improved therapeutic approaches are in constant demand. Although chemotherapy and radiation treatments have assisted in suppressing the growth of tumors, their poor selectivity and efficacy are major limitations for effective therapy en route towards the development of a cure for the cancer epidemic. With the mission of conquering cancer at heart, researchers have pursued a new form of cancer therapy, aptly named, a cancer targeting approach. This method revolves around the selection of a suitable biomarker, typically a cell surface receptor …

Determining The Binding Between Saga Subunits And Spliceosomal Components, Peyton J. Spreacker, Rachel L. Stegeman, Vikki M. Weake

The Summer Undergraduate Research Fellowship (SURF) Symposium

Proper gene regulation is vital to the health and development of an organism. Determining the relationship between splicing, transcription, and chromatin structure is vital for understanding gene regulation as a whole. There have been previous studies linking these elements pairwise; however, no evidence exists for a direct link between all three. Recent data shows that splicing components of the U2 small nuclear ribonucleic protein (snRNP) co-purify with Spt-Ada-Gcn5-acetyltransferase (SAGA), a highly conserved transcriptional co-activator and chromatin modifier. We hypothesize that SAGA binds with splicing components through a multi-protein binding surface with certain core components based on preliminary yeast two-hybrid data. …

Antiviral Responses In Mouse Embryonic Stem Cells: Differential Development Of Cellular Mechanisms In Type I Interferon Production And Response, Ruoxing Wang

Dissertations

Embryonic stem cells (ESCs) have been recognized as a promising cell source for regenerative medicine. Intensive research over the past decade has led to the possibility that ESC-derived cells will be used for the treatment of human diseases. However, increasing evidence indicates that ESC-derived cells generated by the current differentiation methods are not fully functional. It is recently recognized that ESC-derived cells lack innate immunity to a wide range of infectious agents and inflammatory cytokines. When used in patients, ESC-derived cells would be placed in wounded sites that are exposed to various pathogens and inflammatory cytokines; therefore, their viability and …

Characterizing Populations Of Non-Coding Rnas In Karenia Brevis At Different Times Of The Diel Cycle, Scott Boyd Anglin

Master's Theses

Karenia brevis is a mixotrophic, marine dinoflagellate found in the Gulf of Mexico that generates periodic, if not annual, harmful algal blooms (also known as “red tides”) in certain coastal areas. In an effort to better understand the biology of this organism, a functional genomics project has been initiated. As part of that project, it has been determined that a significant number of natural antisense transcripts (NATs) as well as double-stranded RNA (dsRNA) molecules exist within the transcriptome of K. brevis. I hypothesize that the non-coding NATs, similar to microRNAs (miRNAs) in other organisms play a role in regulating …

Exploration Into The Cellular Effects Of Aziridinomitosenes, Christopher M. Mallory

Boise State University Theses and Dissertations

Aziridinomitosenes (AZMs) are organic compounds structurally related to the mitomycins, a class of anti-tumor agents and antibiotics. The cytotoxicity of the mitomycins is correlated to their ability to covalently link complimentary strands of DNA, forming DNA interstrand cross-links (ICLs). Currently, there has been limited investigation into the biological activity of AZMs, likely due to difficulties in their synthesis. Our lab has synthesized and evaluated the cellular effects of two AZMs, (1S, 2S)-6-desmethyl(methylaziridino)mitosene (H/H-AZM) and (1S, 2S)-6-methyl(methylaziridino)mitosene (Me/H-AZM). We hypothesize that AZMs exhibit their cytotoxicity and cellular effects following a similar pathway to that of mitomycin C (MC) including …

Biophysical And Computational Investigations Into G-Quadruplex Structural Polymorphism And Interaction With Small Molecules., Huy Tuan Le

Electronic Theses and Dissertations

In the cell, guanine-rich nucleic acids can self-assemble into unique four stranded tertiary structures known as G-quadruplexes. G-quadruplex formation in the telomere leads inhibits telomerase, an enzyme activated in cancer cells to maintain the telomere and allowing for cancer cells to achieve immortality. G-quadruplex formation in the promoters and 5’-untranslated regions regulates the expression of many oncogenes. Furthermore, G-quadruplex formation during cellular replication promotes genomic instability, a characteristic which enables tumor development. Because of their implication in cancer, G-quadruplex structures have emerged as attractive drug targets for anti-tumor therapeutics. In the current dissertation work, we present three experimental approaches to …

Pi3k- And Mtor-Dependent Mechanisms Of Lapatinib Resistance And Resulting Therapeutic Opportunities, Samuel Brady

Dissertations and Theses (Open Access)

Breast cancers with HER2 amplification represent 20-25% of breast cancer cases and are frequently responsive to the HER2 kinase inhibitor lapatinib, but generally for only short duration. We aimed to understand how breast cancers with HER2 amplification become resistant to lapatinib, in order to identify potential therapies that can overcome lapatinib resistance. To establish lapatinib resistance models we treated three HER2+ breast cancer cell lines with lapatinib for several months until they became lapatinib-resistant. We then compared lapatinib-sensitive (parental) cells with their lapatinib-resistant (LapR) counterparts to identify changes conferring lapatinib resistance. We found that activation of PI3K, specifically the p110α …

Genetic And Mechanistic Analysis Of Rat Mammary Cancer Susceptibility., Jennifer Sanders

Electronic Theses and Dissertations

Breast cancer is a complex disease, which is influenced by genetic, epigenetic and environmental components. Genetic susceptibility to breast cancer is made up of high, moderate and low penetrance alleles. High and moderate penetrance alleles are rare and constitute only a small percentage of the genetic susceptibility. Most variation in genetic susceptibility is controlled by low- penetrance, common polymorphisms. Comparative genetics uses model organisms to study human disease. Rat strains exhibit different susceptibility phenotypes to chemical induced carcinogenesis. The Wistar-Furth (WF) rat strain is susceptible to chemically induced mammary carcinogenesis, while the Wistar-Kyoto (WKy) and Copenhagen (COP) rat strains are …

Roles For B-Raf Kinase In The Specific Regulation Of Α4Β1 Integrin In T Cells, Wells S. Brown

Dissertations and Theses (Open Access)

The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to ECM or other cells, as well as initiating intracellular signaling cascades promoting survival or activation. The mitogen activated-protein kinase (MAPK) pathway is known to be downstream from integrins and regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show chemical inhibition of RAF by Sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion …

Egfr Modulates Microrna Maturation In Response To Hypoxia Through Phosphorylation Of Argonaute2, Jia Shen

Dissertations and Theses (Open Access)

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at posttranscriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in regulatory roles to cope with a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumor. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumor cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here, we show that EGFR, …

Energy Stress Causes Chaperones To Assemble Into Cytoplasmic Complexes, Kimberly J. Cope

Dissertations and Theses (Open Access)

The majority of proteins require molecular chaperones to assist their folding into tertiary and quaternary structures. Certain stresses can compromise the weak hydrophobic forces responsible for these structures and lead to protein unfolding, misfolding, and aggregation. Aggregates of proteins are hallmarks of devastating diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. Fortunately, bacteria, plants, and fungi have a potent disaggregase, named Hsp104 in Saccharomyces cerevisiae. Recently, heat-induced aggregates, termed Q-bodies, were found to contain three molecular chaperones: Hsp70, Hsp104, and Hsp42. Their coalescence from small puncta into larger inclusions required Hsp104. During glucose deprivation, a stress that isn’t known to …

Characterization Of A Putative Sir2 Like Deacetylase And Its Role In Sabp2 Dependent Salicylic Acid Mediated Pathways In Plant, Md I. Haq

Electronic Theses and Dissertations

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant …

Some Effects Of The Human Genome Project On The Erdős Collaboration Graph, Chris Fields

Journal of Humanistic Mathematics

The Human Genome Project introduced large-scale collaborations involving dozens to hundreds of scientists into biology. It also created a pressing need to solve discrete mathematics problems involving tens of thousands of elements. In this paper, we use minimal path lengths in the Erdős Collaboration Graph between prominent individual researchers as a measure of the distance between disciplines, and we show that the Human Genome Project brought laboratory biology as a whole closer to mathematics. We also define a novel graph reduction method and a metric that emphasizes the robustness of collaborative connections between researchers; these can facilitate the analysis of …

Key Residues Of Human Cytoplasmic Protein Tyrosine Phosphatase-A And -B For Substrate Binding And Specificity, Byunghyun Park

Open Access Theses

Reversible tyrosine phosphorylation plays an important role in signaling pathways that are essential for regulating cellular growth, differentiation and metabolism. Moreover, several human diseases such as diabetes, obesity and cancers are associated with the deregulation of protein tyrosine phosphatases (PTPs). Several studies provide evidence that PTPs not only contribute to cellular differentiation, but over-expression of these molecules also leads to transformation of non-transfomed cells as well. Based on these results, designing specific PTP inhibitors may ultimately function as potential therapeutic agents to treat various diseases including cancer, diabetes, and autoimmune diseases. EphA2 is a receptor tyrosine kinase which is hypo-phosphorylated …