Biotechnology Commons^™

Development Of In Vivo Systems For Detecting And Studying Ribosome Inhibition By Small Molecules, Shijie Huang

Chemistry and Chemical Biology ETDs

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome controlled green fluorescent protein reporter that produce fluorescent signal when the O-ribosome is inhibited. As a proof …

The Prevalence Of Streptococcus Pyogenes And Its Emm Gene Types Among School Children In Ai Ain, Uae, Fatima Hammad Ali Al Shamisi

Theses

Streptococcus pyogenes, also known as Group a streptococcus (GAS), causes a wide variety of diseases in children and adults, and it frequently infects the pharynx and skin; pharyngitis and tonsillitis are one of the commonest diseases caused by this pathogen. GAS evaded by many virulence factors, however, the most significant part is the M protein, which plays a major role in its pathogenicity. Over 225 M protein types have been identified so far by the emm typing technique. Identification and emm typing of GAS is an essential part of epidemiological and pathogenic studies of streptococcal diseases, as different regions of …

Microarray Analysis Of Arabidopsis Wrky33 Mutants In Response To The Necrotrophic Fungus Botrytis Cinerea, Shamma Saeed Salem Al Shamsi

Theses

Plants are exposed to many environmental stresses that affect their growth and development. These stresses include biotic stresses (via organisms) and abiotic stresses (via environment). Plants respond to these stresses by transcriptional reprogramming and different signaling pathways. Arabidopsis thaliana has shown great sensitivity to the biotic stress Botrytis cinerea. The WRKY33 gene plays an important role in plant defense mechanism against this pathogen. The overall goal is to identify common regulated genes of wrky33 mutant and 35S:WRKY33 over expressing transgenic lines in response to B. cinerea; ultimately to improve plant stress tolerance in Arabidopsis. The specific aims are to: (1) …

Assessing The Risk Of Resistance Evolution, Adult And Larval Susceptibility, And Sublethal Effects After Exposure Of Corn Rootworms To Vacuolar Atpase-A And Snf7 Dsrnas, Adriano Elias Pereira

Department of Entomology: Dissertations, Theses, and Student Research

Corn rootworms are Diabroticite beetles from the genus Diabrotica and include the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, and the northern corn rootworm, D. barberi Smith & Lawrence, as the most important pests in the U.S. Corn Belt. The southern corn rootworm (SCR), D. undecimpunctata howardi Barber, is considered a polyphagous plant pest and feeds in a wide variety of plants including peanuts, cucurbits, soybeans, and especially corn. Resistance to control methods including behavioral resistance to crop rotation and to corn hybrids expressing Bacillus thuringiensis Berliner toxins in WCR has frequently been reported. SCR has been considered a …

Large-Scale Off-Target Identification Using Fast And Accurate Dual Regularized One- Class Collaborative Filtering And Its Application To Drug Repurposing, Hansaim Lim, Aleksandar Poleksic, Yuan Yao, Hanghang Tong, Di He, Luke Zhuang, Patrick Meng, Lei Xie

Publications and Research

Target-based screening is one of the major approaches in drug discovery. Besides the intended target, unexpected drug off-target interactions often occur, and many of them have not been recognized and characterized. The off-target interactions can be responsible for either therapeutic or side effects. Thus, identifying the genome-wide off-targets of lead compounds or existing drugs will be critical for designing effective and safe drugs, and providing new opportunities for drug repurposing. Although many computational methods have been developed to predict drug-target interactions, they are either less accurate than the one that we are proposing here or computationally too intensive, thereby limiting …

Microfluidic Cantilever Detects Bacteria And Measures Their Susceptibility To Antibiotics In Small Confined Volumes, Hashem Etayash, M. F. Khan, Kamaljit Kaur, Thomas Thundat

Pharmacy Faculty Articles and Research

In the fight against drug-resistant bacteria, accurate and high-throughput detection is essential. Here, a bimaterial microcantilever with an embedded microfluidic channel with internal surfaces chemically or physically functionalized with receptors selectively captures the bacteria passing through the channel. Bacterial adsorption inside the cantilever results in changes in the resonance frequency (mass) and cantilever deflection (adsorption stress). The excitation of trapped bacteria using infrared radiation (IR) causes the cantilever to deflect in proportion to the infrared absorption of the bacteria, providing a nanomechanical infrared spectrum for selective identification. We demonstrate the in situ detection and discrimination of Listeria monocytogenes at a …

Structural Determinants Allowing Transferase Activity In Sensitive To Freezing 2, Classified As A Family I Glycosyl Hydrolase, Rebecca Roston, Kun Wang, Leslie A. Kuhn, Christoph Benning

Department of Biochemistry: Faculty Publications

Background: SENSITIVE TO FREEZING 2 (SFR2) is classified as a glycosyl hydrolase, and by using glycosyltransferase activity, it modifies membrane lipids to promote freeze tolerance.

Results: Although the active site of SFR2 is identical to hydrolases, adjacent loop regions contribute to its transferase activity.

Conclusion: Transferase activity evolved by modifications external to the core catalytic site.

Significance: Defined structure-function relationships will inform engineering of transferases and freeze tolerance.

Sending Out An Sos: Mitochondria As A Signaling Hub, Iryna Bohovych, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Normal cellular physiology is critically dependent on numerous mitochondrial activities including energy conversion, cofactor and precursor metabolite synthesis, and regulation of ion and redox homeostasis. Advances in mitochondrial research during the last two decades provide solid evidence that these organelles are deeply integrated with the rest of the cell and multiple mechanisms are in place to monitor and communicate functional states of mitochondria. In many cases, however, the exact molecular nature of various mitochondria-to-cell communication pathways is only beginning to emerge. Here, we review various signals emitted by distressed or dysfunctional mitochondria and the stress-responsive pathways activated in response to …

Optimizing Gdna Purification And Qpcr Techniques For Specific Detection And Enumeration Of Probiotic Bacteria, Katelynn C. Lee, Jennifer Vanderkelen, Christopher Kitts

STAR Program Research Presentations

Probiotics are live microorganisms, like bacteria and yeast, which are thought to provide health benefits when ingested. Large manufacturers produce blends of probiotics for supplementing the diets of agriculturally important animals. Commercial probiotic products are labeled with the number of cells present at the time of manufacture. Our assignment was to develop an assay which can be used to quantify the number of specific lactic acid bacteria present in certain probiotic products, thus verifying the number reported on the product labels. This quantification process involves isolating genomic DNA (gDNA) from the given samples and then running the DNA through a …

Contribution Of A Putative Up Element Dna Sequence To The Activity Of A Newly Identified Phage Promoter, Courtney Hamilton

Mahurin Honors College Capstone Experience/Thesis Projects

In transcription, a universal step in gene expression, information from a DNA sequence is copied into RNA. A key component in gene expression is the promoter sequence, a region of DNA to which RNA polymerase binds during the initiation of transcription of downstream genes. Most bacterial promoters contain a -10 and a -35 sequence that are bound by the RNA polymerase. Some promoters also contain an Upstream Promoter (UP) element. UP elements have been shown to boost promoter activity. We recently identified a new promoter in a mutant bacteriophage that grows on a bacterial host that prevents antitermination of phage …

Design And Development Of A Plasmid Vector For Protein Expression And Purification, Mahima Grover, Craig Sweet, David H. Thompson

The Summer Undergraduate Research Fellowship (SURF) Symposium

Production and isolation of proteins are difficult, costly and time-consuming processes. The aim of this project is for the development of plasmids, which allow for streamlined production and isolation of proteins. To allow for modular insertion of varying segments of DNA we are using ‘recursive directional ligation by plasmid reconstruction’. This technique uses type II restriction endonucleases, which cut downstream from their recognition site allowing multiple insertions without losing a restriction site. Using this process, we can ligate multiple DNA sequences together and express them to be able to construct a scar less fusion protein. In order to accomplish this, …

The Effect Of An Enhanced Isopentenyl Monophosphate Pool On Terpenoid Biosynthesis In Vivo, Evan T. Adams, Laura K. Henry, Dr. Natalia Dudareva

The Summer Undergraduate Research Fellowship (SURF) Symposium

Found in all living organisms, terpenoids make up the largest group of natural products and are essential compounds for many major processes, including photosynthesis, respiration, hormone production, and electron transport. Additionally, they have commercial and medical value in products including fragrances, cosmetics, and medicines. Terpenoids originate from the five-carbon building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized by the mevalonic acid (MVA) and methylerithritol phosphate (MEP) pathways. An alternative MVA pathway was discovered in Archaea with the final two enzymes being phosphomevalonate decarboxylase (MPD) and isopentenyl phosphate kinase (IPK). Even though this alternative pathway is not …

Optimization Of A Genomic Editing System Using Crispr/Cas9-Induced Site-Specific Gene Integration, Jillian L. Mccool Ms., Nick Hum, Gabriela G. Loots

STAR Program Research Presentations

The CRISPR-Cas system is an adaptive immune system found in bacteria which helps protect against the invasion of other microorganisms. This system induces double stranded breaks at precise genomic loci (1) in which repairs are initiated and insertions of a target are completed in the process. This mechanism can be used in eukaryotic cells in combination with sgRNAs (1) as a tool for genome editing. By using this CRISPR-Cas system, in addition to the “safe harbor locus,” ROSAβ26, the incorporation of a target gene into a site that is not susceptible to gene silencing effects can be achieved through few …

Vascular Endothelial Growth Factor-A Gene Electrotransfer Promotes Angiogenesis In A Porcine Model Of Cardiac Ischemia, Anna A. Bulysheva, Barbara Hargrave, Nina Burcus, Cathryn G. Lundberg, Len Murray, Richard Heller

Bioelectrics Publications

This study aimed to assess safety and therapeutic potential of gene electrotransfer (GET) as a method for delivery of plasmid encoding vascular endothelial growth factor A (VEGF-A) to ischemic myocardium in a porcine model. Myocardial ischemia was induced by surgically occluding the left anterior descending coronary artery in swine. GET following plasmid encoding VEGF-A injection was performed at four sites in the ischemic region. Control groups either received injections of the plasmid without electrotransfer or injections of the saline vehicle. Animals were monitored for 7 weeks and the hearts were evaluated for angiogenesis, myocardial infarct size and left ventricular contractility. …

Loss Of Exogenous Androgen Dependence By Prostate Tumor Cells Is Associated With Elevated Glucuronidation Potential, Brenna M. Zimmer, Michelle E. Howell, Qin Wei, Linlin Ma, Trevor Romsdahl, Eileen G. Loughman, Jennifer E. Markham, Javier Seravalli, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD⁺-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate, and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen dependent versus castration resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration resistant tumor cells expressed higher …

Novel Mechanisms Of Β-Adrenergic Signaling In Prostate Cancer Progression, Mohit Hulsurkar

Dissertations and Theses (Open Access)

Prostate cancer is the second leading cause of cancer death among American men. The American Cancer Society estimates that 180,890 men will be will be diagnosed with prostate cancer in 2016 in the USA. (http://www.cancer.org/cancer/prostatecancer/detailedguide/prostate-cancer-key-statistics). Androgen deprivation therapy (ADT) is the standard treatment for early stage prostate cancer. But most patients relapse with aggressive variants of prostate cancer, with survival time between 1-3 years. In order to develop cure for such aggressive variants of prostate cancer, our present understanding of the mechanisms underlying its progression needs to be advanced.

Recently, it has been found that activation of β-adrenergic signaling pathway …

Assessing Stress Responses To Atmospheric Cold Plasma Exposure Using Escherichia Coli Knock Out Mutants, Lu Han, Daniela Boehm, Sonal Patil, Patrick J. Cullen, Paula Bourke

Articles

Aim: This study investigated the effect of Atmospheric cold plasma (ACP) exposure induced stress on microbial inactivation patterns and the regulation of genes involved in the microbial stress response in conjunction with key processing parameters of exposure time and post treatment storage time.

Methods and Results: Cell suspensions of Escherichia coli BW 25113 and its isogenic knock-out mutants in rpoS, soxR, soxS, oxyR and dnaK genes were treated with high voltage ACP in a sealed package for 1, 3 and 5 min followed by 0, 1 and 24 h post-treatment storage. ROS densities and colony formation were …

Microbubble Generation By Piezoelectric Transducers For Biomedical Studies, Mohammed Alkhazal

Electrical & Computer Engineering Theses & Dissertations

Bubbles induced by blast waves or shocks are speculated as the major cause of damage in biological cells in mild traumatic brain injuries (TBI). Microbubble collapse was found to induce noticeable cell detachment from the cell substrate, changes in focal adhesion, and biomechanics. To better understand the bubble mechanism, a system needs to be constructed which allows clear differentiation on the impact of bubbles from that of shocks. Such a generator needs to be low profile in order to place under a microscope. A piezoelectric transducer system was designed to meet the need. The system uses either a flat or …

Small-Angle X-Ray Scattering Studies Of The Oligomeric State And Quaternary Structure Of The Trifunctional Proline Utilization A (Puta) Flavoprotein From Escherichia Coli, Ranjan K. Singh, John D. Larson, Weidong Zhu, Robert P. Rambo, Greg L. Hura, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Background: Trifunctional proline utilization A (PutA) proteins are multifunctional flavoproteins that catalyze two reactions and repress transcription of the put regulon.

Results: PutA from Escherichia coli is a V-shaped dimer, with the DNA-binding domain mediating dimerization.

Conclusion: Oligomeric state and quaternary structures are not conserved by PutAs.

Significance: The first three-dimensional structural information for any trifunctional PutA is reported.

Effect Of Rapamycin As An Inhibitor Of The Mtor Cell Cycle Entry Complex On The Selective Lysis Of Human Leukemia Cells Lines In Vitro Using 20 Khz Pulsed Low-Frequency Ultrasound, Yasmine Hemida

Renée Crown University Honors Thesis Projects - All

It has been shown that the mammalian target of rapamycin (mTOR) pathway, which regulates cell growth and proliferation, is aberrant in many hematological malignancies. Rapamycin inhibits mTOR signaling which regulates cell growth and cell cycle progression. This study sought to determine the effects of mTOR inhibitors on the cell sizes of normal and neoplastic cells and to determine ultrasonic sensitivity of normal and neoplastic cells treated with mTOR inhibitors. The effects of rapamycin (rapa), an extensively studied natural product that affects cell cycle entry by inhibiting mTORC1, and its analogs temsirolimus (tems) and everolimus (eve), were examined on leukemic cell …