Biochemistry, Biophysics, and Structural Biology Commons^™

A Mechanistic Study Of An Ipsc Model For Leigh’S Disease Caused By Mtdna Mutataion (8993 T>G), John P. Galdun

Theses and Dissertations

Mitochondrial diseases encompass a broad range of devastating disorders that typically affect tissues with high-energy requirements. These disorders have been difficult to diagnose and research because of the complexity of mitochondrial genetics, and the large variability seen among patient populations. We have devised and carried out a mechanistic study to generate a cell based model for Leigh’s disease caused by mitochondrial DNA mutation 8993 T>G. Leigh’s disease is a multi-organ system disorder that depends heavily on the mutation burden seen within various tissues. Using new reprogramming and sequencing technologies, we were able to show that Leigh’s disease patient fibroblasts …

Investigation Of Anaplasma Phagocytophilum And Anaplasma Marginale Adhesin-Host Cell Interactions, Kathryn S. Hebert

Theses and Dissertations

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and …

A Pipeline For Creation Of Genome-Scale Metabolic Reconstructions, Shaun W. Norris

Theses and Dissertations

The decreasing costs of next generation sequencing technologies and the increasing speeds at which they work have lead to an abundance of 'omic datasets. The need for tools and methods to analyze, annotate, and model these datasets to better understand biological systems is growing. Here we present a novel software pipeline to reconstruct the metabolic model of an organism in silico starting from its genome sequence and a novel compilation of biological databases to better serve the generation of metabolic models. We validate these methods using five Gardnerella vaginalis strains and compare the gene annotation results to NCBI and the …

Reduction Of Oxidative Stress And Storage Lesions (Rcsl) In Red Blood Cells - Analysis Of Ascorbic Acid (Aa), N-Acetylcysteine Amide (Ad4), And Serotonin (5-Ht), Shanmuka Gadiraju

Undergraduate Research Posters

Oxidative stress is a common occurrence in red blood cell (RBC) storage in blood banks throughout the world. Typically RBC units stored under routine standard protocol (stored in SAGM-CPD additive solution) can only be kept up to 42 days for transfusion usage before being discarded. I am studying the effects of Ascorbic Acid (AA), N-acetylcysteine amide (AD4), and Serotonin (5-HT) as additives in blood bank storage because I want to find out if these additives can reduce storage-induced oxidative stress on red blood cells (RBCs), in order to help my reader understand how potential blood storage additives can affect the …

Probing Allosteric, Partial Inhibition Of Thrombin Using Novel Anticoagulants, Stephen S. Verespy Iii

Theses and Dissertations

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an …

High Affinity Block Of Icl,Swell By Thiol-Reactive Small Molecules, Sung H. Park

Theses and Dissertations

Ebselen (Ebs) is considered as a glutathione peroxidase (GPx) mimetic and primarily thought to function by scavenging intracellular reactive oxygen species (ROS). Previous to our work, Deng et al. (2010a) demonstrated complete block of I_Cl,swell with 15 microM Ebs following endothelin-1 (ET-1) induced activation of the current in cardiomyocytes. This block was presumed to take effect mainly via the quenching of ROS. Nonetheless, our work with DI TNC1 astrocytes strongly emphasizes that Ebs might function by an alternative mechanism based on its kinetic profile in blocking I_Cl,swell. Our experiments showed that 45 nM Ebs can fully block …

Investigating The Mechanism Of The Compartmentalized Cbp (Creb-Binding Protein) Ubiquitin Ligase Activities, Oluwatoyin E. Akande

Theses and Dissertations

CBP (CREB Binding Protein) is global transcriptional co-activator and histone acetyltransferase. CBP is involved in the modulation of the transcription of many genes via histone acetylation at the promoter regions of the target genes. Also, non-histone proteins and transcription factors can be acetylated by CBP to promote their transcriptional activation. In addition to its transcription co-activator role, CBP is involved in many other pathological and physiological cellular processes such as cell growth and differentiation, cell transformation and development, response to stress, cell cycle regulation and apoptosis.

CBP and its paralogue p300, play double-edged roles in the regulation of p53, a …

Celecoxib Enhances Sorafenib/Sildenafil Lethality In Cancer Cells And Reverts Platinum Chemotherapy Resistance, Timothy A. Webb

Theses and Dissertations

The present studies sought to determine whether the lethality of the drug combination [sorafenib + sildenafil] could be enhanced by the anti-inflammatory agent celecoxib, using ovarian cancer and other tumor cell lines as models. Also, in a dose dependent fashion celecoxib enhanced [sorafenib + sildenafil] lethality in multiple ovarian cancer cell lines. In a dose dependent fashion celecoxib enhanced the ability of [sorafenib + sildenafil] to reduce expression of multiple chaperone proteins in parallel with lower levels of the drug efflux pumps ABCB1 and ABCG2. Over-expression of GRP78 and HSP27 maintained pump expression in the presence of drugs. Cell killing …

The Study Of The Regulon Of Oxyr In Escherichia Coli And Porphyromonas Gingivalis, Christopher K. Pham

Theses and Dissertations

The facultative anaerobe, Escherichia coli and the obligate anaerobe, Porphyromonas gingivalis are two bacteria that reside in our body. Although they reside in separate environments, they are both subject to hydrogen peroxide stress and have mechanisms to regulate the stress. OxyR is the primary transcriptional regulator/sensor of oxidative stress response caused by hydrogen peroxide. OxyR in P. gingivalis is not well-characterized compared to OxyR in E. coli. We sought to characterize and compare the two forms of OxyR in order to gain a better understanding of the protein. We determined the oligomeric state of both proteins: primarily a tetramer for …

Resolution Of Inflammation Rescues Axon Initial Segment Disruption, Nicholas M. George

Theses and Dissertations

Axonal domains are required for proper neuron function. These domains are unstable and degenerate concurrent with the inflammation in multiple sclerosis (MS) and the inflammatory disease models experimental autoimmune encephalomyelitis (EAE) and lipopolysaccharide (LPS) induced inflammation. Previous studies from our laboratory have shown that the axon initial segment (AIS) is maintained independently of the presence of myelin, but that AIS disruption is seen in MS as well as EAE and LPS-mediated inflammation. AIS loss can be interrupted in the early stage of EAE using the anti-inflammatory drug Didox. However, the potential for Didox directed repair of the AIS in later …

Conditional Cardiac-Specific Akap13 Knockout Induces Sex Dependent Biventricular Dilated Cardiomyopathy With Sarcomeric And Mitochondrial Defects, Kimberlyn M. Baig-Ward

Theses and Dissertations

Heart disease is a complex and heterogeneous disease. Notably, studies have demonstrated gender differences in the expression and types of cardiovascular disease, such as dilated cardiomyopathy (DCM), a major underlying cause of heart failure. Previously we showed that loss of A-Kinase Anchoring Protein 13 (Akap13), a unique proto-oncogene and estrogen receptor modulator, resulted in enlarged embryonic hearts, defective cardiac sarcomere formation, and embryonic lethality in mice. Data have also shown cAMP-dependent Protein Kinase A (PKA) to be involved in DCM pathophysiology. Given the established role of AKAP13 in cell signaling, its ability to bind and modulate ligand-activated nuclear hormone …

Role Of Vav2 In Podocyte Inflammasome Activation And Glomerular Injury During Hyperhomocysteinemia, Sabena Conley

Theses and Dissertations

Hyperhomocysteinemia (hHcys) is a widely known pathogenic factor in the progression of end-stage renal disease (ESRD) and it is also associated with an increased risk for injurious cardiovascular pathologies during ESRD. HHcys is linked to the formation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, characterized as a critical early mechanism initiating the inflammatory response. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) mediate the activation of the NLRP3 inflammasome in podocytes in response to elevated levels of homocysteine (Hcys) in vitro and in vivo. However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The …

Atm Phosphorylates Subunit A Of Pp2a Resulting In Its Nuclear Export And Spatiotemporal Regulation Of The Dna Damage Response, Amrita D. Sule

Theses and Dissertations

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and major regulator of the DNA damage response (DDR). One critical ATM target is protein phosphatase 2A (PP2A) known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylation of many proteins during the recovery from the DDR while returning the cell to normalcy. Interestingly, ATM and PP2A are known to form an auto-regulatory yin-yang kinase-phosphatase relationship. Herein, we show that the phosphorylation of the PP2A-Aα structural subunit at S401 by ATM results in nuclear export, which regulates the DDR at multiple levels and affects genomic …

From Seed To Sky: Impacts Of Explosive Compounds On Vegetation Across Spatial And Developmental Scales, Stephen M. Via

Theses and Dissertations

Explosive compounds are broadly distributed across the globe as a result of nearly two centuries of munitions use in warfare and military activities. Two explosive compounds have seen disproportionate use; RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and TNT (2-methyl- 1,3,5-trinitrobenzene), being the most commonly found explosives in the environment. The effects of explosives on biota have been studied in great detail; however, there is a general lack of understanding with regard to broader ecological impacts of these contaminants. My dissertation objective was to follow the impacts of explosive compounds on vegetation across scales. Impacts on vegetation at the species scale alter community composition via …

Elucidation Of A Novel Pathway In Staphylococcus Aureus: The Essential Site-Specific Processing Of Ribosomal Protein L27, Erin A. Wall

Theses and Dissertations

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are …

Regulation Of Tie2 Extracellular Complex Formation In Angiogenesis, Annamarie Dalton

Theses and Dissertations

Pathological angiogenesis is an essential component of tumor growth, development, and metastasis for which few effective therapeutic options exist. Though many cancer therapies target the function of cell surface receptors, mechanisms regulating membrane receptor crosstalk remain unclear. Two important families of receptors in angiogenesis, the Ties and Integrins, respond to the extracellular environment via outside-in and, in the case of Integrins, also inside- out signaling. Recent reports showed that the endothelial specific tyrosine kinase receptor, Tie2, forms complexes with two of the endothelial Integrin heterodimers, α₅β₁ and α_Vβ₃, providing a convenient mechanism for …

Mysteries Of The Trypanosomatid Maxicircles: Characterization Of The Maxicircle Genomes And The Evolution Of Rna Editing In The Order Kinetoplastida, Preethi Ranganathan Iyengar

Theses and Dissertations

The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates.

This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 …

Characterization Of A Novel Protease In Staphylococcus Aureus, Adam L. Johnson

Theses and Dissertations

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms …

Ex Vivo Dna Cloning, Adam B. Fisher

Theses and Dissertations

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are …

Functional Characterization Of Rai1 In Zebrafish, Joshua S. Beach

Theses and Dissertations

Smith-Magenis Syndrome (SMS; OMIM #182290) is a multiple congenital abnormality and intellectual disability (ID) disorder caused by either an interstitial deletion of the 17p11.2 region containing the retinoic acid induced-1 (RAI1) gene or a mutation of the RAI1 gene. Individuals diagnosed with SMS typically present characteristics such as ID, self-injurious behavior, sleep disturbance, ocular and otolaryngological abnormalities, craniofacial and skeletal abnormalities, neurological and behavioral abnormalities, as well as other systemic defects and manifestations. Previous work by Vyas in 2009 showed temporal expression of rai1 in zebrafish embryos as early as 9 hpf. We hypothesize that there is maternal …

Rheb Dynamics On Lysosomal Membranes Determines Mtorc1 Activity After Loss Of P53 Or Activation Of Ampk, Catherine M. Bell

Theses and Dissertations

The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss …

Modeling Atp-Binding Cassette G2 (Abcg2) Substrate Specificity, Raghav D. Acharya, Aurijit Sarkar, Glen E. Kellogg

Undergraduate Research Posters

How well can we predict efflux by ATP-binding cassette G2?

It is estimated that there will be about 1.6 million new cases of cancer and half a million cancer deaths in the US during 2015.ATP-binding cassette (ABC) efflux transporters such as ABCG2 are overexpressed in chemotherapy-resistant cancer cells. Anticancer drugs are prone to efflux by these transporters. Being able to identify drugs that are effluxed is of great interest in drug discovery.The current arsenal of methods used to detect efflux are not easily adaptable to high throughput formats and are limited in scope, making experimental analysis an expensive prospect. Hence, …

Dysregulation Of Micrornas In Blood As Biomarkers For Diagnosing Prostate Cancer, Rhonda W. Daniel

Theses and Dissertations

Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers.

Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such …

Hif-Independent Responses In Hypoxia, Divya Padmanabha

Theses and Dissertations

The adaptive response to hypoxia is accompanied by widespread transcriptional changes that allow for prolonged survival in low oxygen. Many of these changes are directly regulated by the conserved hypoxia-inducible factor-1 (HIF-1) complex; however, even in its absence, many oxygen-sensitive transcripts in Caenorhabditis elegans are appropriately regulated in hypoxia. To identify mediators of these non-HIF-dependent responses, I established a hif-1 mutant reporter line that expresses GFP in hypoxia or when worms are treated with the hypoxia mimetic cobalt chloride (cobalt chloride). The reporter is selective and HIF-independent, in that it remains insensitive to a number of cellular stresses, but is …

The Role Of Srsf3 In Control Of Alternative Splicing Of Cpeb2 In Triple Negative Breast Cancer, Brian P. Griffin

Theses and Dissertations

In the presented study, we identified that SRSF3 controls the alternative splicing of CPEB2 and consequently promotes a metastatic phenotype in triple negative breast cancer (TNBC). TNBC causes thousands of deaths annually, frequently due to a lack of effective treatments and a high rate of metastasis in patients. Alternative splicing has been found to be dysregulated in numerous cancers, while splicing factors such as SRSF3 are variably expressed. In this study we performed a siRNA panel to screen potential splicing factors, then used specific siRNA to study the effect of its knockdown on cellular function. These results showed that SRSF3 …

Xlf-Dependent Nonhomologous End Joining Of Complex Dna Double-Strand Breaks With Proximal Thymine Glycol And Screening For Xrcc4-Xlf Interaction Inhibitors, Mohammed Al Mohaini

Theses and Dissertations

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol (Tg) at the first (Tg1) , second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3’ terminus was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate …

Pemetrexed, A Modulator Of Amp-Activated Kinase Signaling And An Inhibitor Of Wild Type And Mutant P53, Stuti Agarwal

Theses and Dissertations

New drug discoveries and new approaches towards diagnosis and treatment have improved cancer therapeutics remarkably. One of the most influential and effective discoveries in the field of cancer therapeutics was antimetabolites, such as the antifolates. The interest in antifolates increased as some of the antifolates showed responses in cancers, such as mesothelioma, leukemia, and breast cancers. When pemetrexed (PTX) was discovered, our laboratory had established that the primary mechanism of action of pemetrexed is to inhibit thymidylate 22 synthase (TS) (E. Taylor et al., 1992). Preclinical studies have shown that PTX has a broad range of antitumor activity in human …

Interaction Between Atm Kinase And P53 In Determining Glioma Radiosensitivity, Syed F. Ahmad

Theses and Dissertations

Glioblastoma multiforme (GBM) is the most common primary brain tumor. Studies have shown that targeting the DNA damage response can sensitize cancer cells to DNA damaging agents. Ataxia telangiectasia mutated (ATM) is involved in signaling DNA double strand breaks. Our group has previously shown that ATM inhibitors (ATMi) sensitize GBM cells and tumors to ionizing radiation. This effect is greater when the tumor suppressor p53 is mutated.

The goals of this work include validation of a new ATM inhibitor, AZ32, and elucidation of how ATMi and p53 status interact to promote cell death after radiation. We propose that ATMi and …

Nitric Oxide Synthase Activity And Its Modulation In The Treatment Of Colorectal Cancer, Asim Alam

Theses and Dissertations

The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under …

Hcpr Of Porphyromonas Gingivalis Utilizes Heme To Bind No, Benjamin Belvin

Theses and Dissertations

The obligate anaerobe Porphyromonas gingivalis is the etiological agent responsible for periodontal disease. It must withstand high levels of reactive nitrogen species in the oral cavity generated by the host and other oral flora. The mechanisms allowing for protection against such stress remain poorly understand. HcpR is an FNR-CRP family regulator that has been implicated in regulation of the nitrosative stress response. In this study we characterize the biochemical properties of HcpR. It is a homo-dimer that is composed of 3 domains – a heme-binding domain, dimerization helix, and a DNA-binding domain. Our studies show that HcpR binds the heme …